Conjugates of synthetic TLR agonists and uses therefor

ABSTRACT

The invention provides TLR agonists and conjugates thereof useful in vaccines and to prevent, inhibit or treat a variety of disorders including pathogen infection and asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of and claims priority under 35 U.S.C.§120 to U.S. patent application Ser. No. 13/736,545, filed Jan. 8, 2013,which application is a divisional of and claims the benefit of priorityunder 35 U.S.C. §120 to U.S. patent application Ser. No. 12/027,960,filed on Feb. 7, 2008, now issued as U.S. Pat. No. 8,357,374, whichapplication claims benefit of the filing date of U.S. application Ser.No. 60/888,699, filed on Feb. 7, 2007, all of which are incorporated byreference herein in their entireties.

STATEMENT OF GOVERNMENT RIGHTS

The invention described herein was made with government support underGrant Number AI056453 awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

BACKGROUND

A great deal has been learned about the molecular basis of innaterecognition of microbial pathogens in the last decade. It is generallyaccepted that many somatic cells express a range of pattern recognitionreceptors that detect potential pathogens independently of the adaptiveimmune system (see Janeway et al., Annu Rev. Immunol., 20:197 (2002)).These receptors are believed to interact with microbial componentstermed pathogen associated molecular patterns (PAMPs). Examples of PAMPsinclude peptidoglycans, lipotechoic acids from gram-positive cell walls,the sugar mannose (which is common in microbial carbohydrates but rarein humans), bacterial DNA, double-stranded RNA from viruses, and glucansfrom fungal cell walls. PAMPs generally meet certain criteria thatinclude, (a) their expression by microbes but not their mammalian hosts,(b) conservation of structure across the wide range of pathogens, and(c) the capacity to stimulate innate immunity. Toll-like Receptors(TLRs) have been found to play a central role in the detection of PAMPsand in the early response to microbial infections (see Underhill et al.,Curr. Opin. Immunol., 14:103 (2002)). Ten mammalian TLRs and a number oftheir agonists have been identified. For example, TLR7 and TLR9recognize and respond to imiquimod and immunostimulatory CpGoligonucleotides (ISS-ODN), respectively. The synthetic immunomodulatorR-848 (resiquimod) activates both TLR7 and TLR8. While TLR stimulationinitiates a common signaling cascade (involving the adaptor proteinMyD88, the transcription factor NF-kB, and pro-inflammatory and effectorcytokines), certain cell types tend to produce certain TLRs. Forexample, TLR7 and TLR9 are found predominantly on the internal faces ofendosomes in dendritic cells (DCs) and B lymphocytes (in humans; mousemacrophages express TLR7 and TLR9). TLR8, on the other hand, is found inhuman blood monocytes (see Hornung et al., J. Immunol., 168:4531(2002)).

Interferons (INFs) are also involved in the efficient induction of animmune response, especially after viral infection (Brassard et al., J.Leukoc. Biol., 71:568 (2002)). However, many viruses produce a varietyof proteins that block interferon production or action at variouslevels. Antagonism of interferon is believed to be part of a generalstrategy to evade innate, as well as adaptive immunity (see Levy et al.,Cytokine Growth Factor Rev., 12:143 (2001)). While TLR agonists may besufficiently active for some methods of treatment, in some instances themicrobial interferon antagonists could mitigate the adjuvant effects ofsynthetic TLR agonist.

A more specific response to microbial infections is based on active orpassive immunization. If universal immunization is not consideredcost-effective (or pharmacoeconomically viable), identification of apopulation at-risk that would benefit from immunoprophylaxis may becost-effective, although identifying that population may be notstraightforward. Nevertheless, there are some clearly defined at-riskpopulations for certain bacterial infections, such as staphylococcalinfections, including dialysis patients, patients withventriculoperitoneal shunts, patients at-risk of infective endocarditis,and residents of nursing homes, all of which suffer from chronicconditions that place them at a prolonged increased risk fromstaphylococcal infections. Many of these patients are also at increasedrisk for acquiring healthcare-associated methicillin-resistantStaphylococcus aureus (HA-MRSA). Blocking colonization ofStaphylococcus, however, may be more achievable than protecting againstinfection.

Passive immunoprophylaxis using either polyclonal antibodies (Capparelliet al., Antimicrob. Agents Chemo., 49:4121 (2005)) or monoclonalantibodies (mAbs) (www.biosynexus.com/productcandidates.html) mayprovide immediate (although short-term) protection for patients whoeither cannot wait for a vaccine effect to occur or whose immune systemsare too compromised to mount a response to a vaccine. One potentialindication for passive immunoprophylaxis is a hospital outbreak ofMRSA-related infections. In such cases, exposed individuals may benefitfrom immediate prophylaxis, whereas individuals residing on the sameward or chronic care facility may benefit from active immunization.Moreover, intensive care unit patients are potential beneficiaries ofpassive immunoprophylaxis, as each of them would likely acquire one ormore risk factors for staphylococcal infections.

SUMMARY OF THE INVENTION

The present invention provides for conjugates of a synthetic TLR agonistlinked via a stable covalent bond to a macromolecule and compositionshaving those conjugates, as well as methods of using the conjugates. Theconjugates may include macromolecules directly linked to a synthetic TLRagonist, e.g., TLR7 or TLR9 agonists, or linked via a linker to the TLRagonist, for instance, linked via an amino group, a carboxy group or asuccinamide group. For instance, the conjugates of the invention includea synthetic TLR agonist (pharmacophore) covalently bound to amacromolecule such as, for instance, a peptide, polypeptide, e.g., anantibody or an antigen binding fragment thereof, lipid, a polymer suchas polyethylene glycol, a bead, such as a polystyrene bead, ordendrimer. The conjugates of the invention are broad-spectrum,long-lasting, and non-toxic synthetic immunostimulatory agents, whichare useful for activating the immune system of a mammal, e.g., a human.In particular, the conjugates of the invention optimize the immuneresponse while limiting undesirable systemic side effects associatedwith unconjugated TLR agonists.

The synthetic TLR agonist may help direct the conjugate to TLRs withinthe endosomes of target cells and enhance delivery of the macromolecule.In one embodiment, the synthetic TLR agonist is specific for endosomalTLRs. In one embodiment, the TLR agonist may be a TLR7, TLR8, TLR3, orTLR9 agonist. Moreover, the synthetic TLR agonist may enhance theresponse to the macromolecule (e.g., immune response). Likewise, themacromolecule may be useful for activating the immune system and/or maydirect the conjugate to particular cells. Thus, the macromolecule, e.g.,one with a primary amino group that is linked to a synthetic TLRagonist, may enhance the activity of the synthetic TLR agonist or have aseparate desirable activity. For example, the macromolecule may enhancethe activity of the TLR agonist by helping to direct the agonist to theTLR within the endosomes of target cells, by enhancing signaltransduction induced by the TLR agonist, or by cross-linking thereceptor, or any combination thereof. In one embodiment, themacromolecule is a lipid which is spontaneously incorporated intoliposomes. In one embodiment, the macromolecule is a nanoparticle whichhas amine groups on its surface. Once coupled to a TLR agonist, the TLRagonist-nanoparticle conjugate may be of a size, for instance, about 100nm, that may reside (be present) in endosomes.

Hospital acquired Staphylococcus aureus (SA) infections are a majorcause of morbidity and mortality. However, vaccines are not used inacute settings because they take too long to act and they are noteffective in immunocompromised patients. The invention provides a methodfor the rapid vaccination of patients at-risk for gram-positivebacterial infections, e.g., SA infections, which employs Toll-likereceptor-7 (TLR7) agonists and one or more antigens (immunogens) of agram-positive bacteria. The use of the vaccines of the invention inducesimmunity in about 6 days, which provides for applications not amenableto standard vaccination protocol (e.g., acute care settings).

As disclosed herein, a composition comprising a gram-positive bacteria,Bacillus anthracis (BA), and a synthetic TLR7 agonist was prepared. Thecomposition induced IL12 and IL6 secretion (indicative of activation ofbone marrow derived macrophages (BMDM)) in vitro and protected miceversus subsequent, otherwise lethal intra-pulmonary BA challenge invivo. In particular, the administration of a composition containing aTLR7 agonist conjugate and an immunogen (UC-IV199-albumin/irradiated BAspores) induced protective immunity to BA within 6 days. In contrast,injection of the animals with BA spores alone, or with BA plus aconventional adjuvant, i.e., cholera toxin (CT), did not protect theanimals from a lethal challenger. The rapidity of a protective immuneresponse in a naïve animal was unexpected.

The invention thus provides immunogenic compositions. In one embodiment,an immunogenic composition of the invention includes a synthetic TLRagonist such as a TLR7 agonist, e.g., UC-IV199, coupled to agram-positive bacterial cell, for instance, coupled to free amino groupson killed Staphylococcus aureus; a synthetic TLR agonist such as a TLR7agonist coupled to a bacterial extract of isolated gram-positivebacterial antigens; a synthetic TLR agonist such as a TLR7 agonistcoupled to isolated gram-positive bacterial protein, e.g., recombinantprotein; or a synthetic TLR agonist such as a TLR7 agonist coupled toisolated gram-positive bacterial carbohydrates. For example, a syntheticTLR7 agonist may be coupled to bacterial carbohydrates using methods toattach Staphylococcus aureus polysaccharides to protein carriers (suchas those used for tetanus toxoid). The killed bacterial preparation maybe prepared using gamma irradiation, heat or chemical treatment. Inanother embodiment, an immunogenic composition of the invention includesa synthetic TLR agonist such as a TLR7 agonist coupled to an adjuvantand a preparation comprising killed gram-positive bacterial cells; asynthetic TLR agonist such as a TLR7 agonist coupled to an adjuvant anda preparation comprising a gram-positive bacterial extract; or asynthetic TLR agonist such as a TLR7 agonist coupled to an adjuvant anda preparation comprising an isolated gram-positive bacterial antigen,e.g., recombinant protein. For example, the immunogenic composition mayinclude UC-IV199 coupled to albumin and a preparation with killedgram-positive bacteria, e.g., killed Staphylococcus aureus. In oneembodiment, an immunogenic composition of the invention includes asynthetic TLR7 agonist coupled to an adjuvant and a preparationcomprising a recombinant gram-positive bacterial antigen, such as anisolated gram-positive bacterial protein or a peptide thereof, orisolated gram-positive bacterial carbohydrate. In one embodiment, asingle dose of the immunogenic composition may show very potentactivity, e.g., provide protective immunity, in a short period of time,e.g., less than about 10 days.

In one embodiment, a sterilized vaccine is administered to a subjectfrom 0 to 7 days prior to hospitalization. In one embodiment, thevaccine is administered intramuscularly. In one embodiment, the vaccineis administered at a dosage between 10 μg and 10 mg.

The use of conjugates of synthetic TLR agonists such as TLR7 agonists ofthe invention is advantageous as accessible and versatile chemistrypermits conjugation to any antigen, and modifiable conjugates havedefined stoichiometry. The conjugates are inexpensive to prepare and arepotent, and so provide rapid protection, enabling use in acute settingssuch as trauma, burn, pre-surgery, or bioterrorism.

Accordingly, there is provided a compound of formula (I):

wherein X¹ is —O—, —S—, or —NR^(c)—;

wherein Y is S or NH;

wherein R^(c) hydrogen, C₁₋₁₀alkyl, or C₁₋₁₀alkyl substituted byC₃₋₆cycloalkyl, or R^(c) and R¹ taken together with the nitrogen atomcan form a heterocyclic ring or a substituted heterocyclic ring, whereinthe substituents are hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkylene,C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkylene, or cyano;

wherein R¹ is (C₁-C₁₀)alkyl, substituted (C₁-C₁₀)alkyl, C₆₋₁₀aryl, orsubstituted C₆₋₁₀aryl, C₅₋₉heterocyclic, substituted C₅₋₉heterocyclic;

wherein each R² is independently hydrogen, —OH, (C₁-C₆)alkyl,substituted (C₁-C₆)alkyl, (C₁-C₆)alkoxy, substituted (C₁-C₆)alkoxy,—C(O)—(C₁-C₆)alkyl(alkanoyl), substituted —C(O)—(C₁-C₆)alkyl,—C(O)—(C₆-C₁₀)aryl(aroyl), substituted —C(O)—(C₆-C₁₀)aryl, —C(O)OH(carboxyl), —C(O)O(C₁-C₆)alkyl(alkoxycarbonyl), substituted—C(O)O(C₁-C₆)alkyl, —NR^(a)R^(b), —C(O)NR^(a)R^(b) (carbamoyl),substituted —C(O)NR^(a)R^(b), halo, nitro, or cyano;

wherein each R^(a) and R^(b) is independently hydrogen, (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkyl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl,aryl, aryl(C₁-C₆)alkyl, Het, Het (C₁-C₆)alkyl, or (C₁-C₆)alkoxycarbonyl;

wherein X² is a bond or a linking group;

wherein R³ is a macromolecule;

wherein n is 1, 2, 3, or 4;

wherein m is 1, 2, 3 or more, e.g., 5, 10, 15, or more;

wherein q is 1, 2, 3 or up to about 1,000, about 10,000 or more, e.g.,about 10⁵, about 10⁶ or greater; or a pharmaceutically acceptable saltthereof. In one embodiment, q is 1 and m is from 1 to 20 or any integerin between. In another embodiment, m is 1 and q is >2. In oneembodiment, m is 1 and R³ may be a virus, e.g., a lentivirus other thansimian immunodeficiency virus (SIV), a retrovirus, an influenza virus, arhinovirus, a papilloma virus, a herpes virus and the like, agram-positive bacterium or bacterial spore, a nanoparticle or a bead,e.g., a silica bead, and q is 10², 10³, 10⁴, 10⁵, 10⁶ or more. Thus, theconjugate may include multimers of the TLR agonist, the macromolecule,or both. The multimers may be linear or branched.

In one embodiment, R³ can be a macromolecule comprising a gram-positivebacteria, peptide of a gram-positive bacterium, protein of agram-positive bacterium, carbohydrate of a gram-positive bacterium, oran adjuvant such as a heterologous protein or peptide, i.e., from asource other than the gram-positive bacteria, such as a host cellprotein or peptide, e.g., albumin or ovalbumin, or a heterologous lipid,heterologous nucleic acid, nanoparticle, bead, such as a polystyrenebead, or dendrimer.

Thus, in various embodiments, m can be 1 or 2; and q can be 1 or 2. Insome embodiments, m is 1 and the surface of the R³ group is linked tohundreds, thousands, or more, q groups. For example, the reactive sitesof a silica particle can be used to link the silica particle to moietiesof a formula described herein. For this configuration, the R³ can be anyother macromolecule disclosed herein, such as a bead, nanoparticle,dendrimer, lipid, spore, or bacterial cell. In other embodiments, theformula and the group R³ can form an alternating chain of three to aboutten repeating groups (e.g., formula-R³-formula-R³-formula-R³—, etc).

The macromolecule in the conjugates of the invention forms a stable bondwith the TLR agonist, i.e., the conjugate does not act as a prodrug. Themacromolecule can include organic molecules, composed of carbon, oxygen,hydrogen, nitrogen, sulfur, phosphorous, or any combination thereof, solong as the macromolecule is not harmful to body tissues (e.g., it isnon-toxic, and/or does not cause inflammation). The macromolecule maypermit targeting or enhance the immune response, e.g., the macromoleculemay be an antigen such as a melanoma-specific peptide.

In various embodiments, when more than one R³ is present in a moleculeof any formula described herein, each R³ may be the same, or the R³groups may be different from each other. Accordingly, when more than oneR³ group is present, each R³ is independently a group as defined foreach formula.

In addition, the invention provides a pharmaceutical compositioncomprising at least one compound of formula (I), or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable diluent or carrier.

The invention includes the use of conjugates of a synthetic TLR agonistand a macromolecule, as well as TLR agonist conjugates and anothermolecule. The conjugate may be useful to prevent, inhibit or treatdisorders including, but not limited to, allergic asthma, infectiousdiseases such as respiratory viral infections, e.g., those associatedwith influenza virus or respiratory syncytial virus (RSV) infection,lupus and other autoimmune diseases, and as a vaccine, e.g., for canceror infectious diseases. In one embodiment, a single dose of theconjugate may show very potent activity in stimulating the immuneresponse. Moreover, because of the low toxicity of the conjugates, insome circumstances higher doses may be administered, e.g., systemically,while under other circumstances lower doses may be administered, e.g.,due to localization of the conjugate. In one embodiment, whenadministered at high doses, the synthetic TLR agonist conjugates mayelicit an antagonistic response, and so may be useful to inhibit ortreat asthma or autoimmune diseases. A first dose may elicit ahyperresponse which, in turn, suppresses the immune response, therebyavoiding inflammation. Thus, the use of higher doses andreadministration may result in inhibition of an immune response.

In one embodiment, the invention provides a method to prevent or inhibita gram-positive bacterial infection in a mammal. The method includesadministering to the mammal an effective amount of a compositioncomprising a bacterial antigen of a gram-positive bacteria and an amountof a compound having formula (IA):

-   wherein X¹ is —O—, —S—, or —NR^(c)—;-   wherein R^(c) is hydrogen, C₁₋₁₀alkyl, or substituted C₁₋₁₀alkyl, or    R^(c) and R¹ taken together with the nitrogen atom can form a    heterocyclic ring or a substituted heterocyclic ring, wherein the    substituents on the alkyl, aryl or heterocyclic groups are hydroxy,    C₁₋₆alkyl, hydroxyC₁₋₆alkylene, C₁₋₆alkoxy, C₃₋₆cycloalkyl,    C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano, halogen, or aryl;-   R¹ is hydrogen, (C₁-C₁₀)alkyl, substituted (C₁-C₁₀)alkyl, C₆₋₁₀aryl,    or substituted C₆₋₁₀aryl, C₅₋₉heterocyclic, substituted    C₅₋₉heterocyclic; wherein the substituents on the alkyl, aryl or    heterocyclic groups are hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkylene,    C₁₋₆alkoxy, C₃₋₆cycloalkyl, C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano,    halogen, or aryl;-   each R² is independently hydrogen, —OH, (C₁-C₆)alkyl, substituted    (C₁-C₆)alkyl, (C₁-C₆)alkoxy, substituted (C₁-C₆)alkoxy,    —C(O)—(C₁-C₆)alkyl(alkanoyl), substituted —C(O)—(C₁-C₆)alkyl,    —C(O)—(C₆-C₁₀)aryl(aroyl), substituted —C(O)—(C₆-C₁₀)aryl, —C(O)OH    (carboxyl), —C(O)O(C₁-C₆)alkyl(alkoxycarbonyl), substituted    —C(O)O(C₁-C₆)alkyl, —NR^(a)R^(b), —C(O)NR^(a)R^(b) (carbamoyl),    substituted —C(O)NR^(a)R^(b), halo, nitro, or cyano; wherein the    substituents on the alkyl, aryl or heterocyclic groups are hydroxy,    C₁₋₆alkyl, hydroxyC₁₋₆alkylene, C₁₋₆alkoxy, C₃₋₆cycloalkyl,    C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano, halogen, or aryl;-   each R^(a) and R^(b) is independently hydrogen, (C₁-C₆)alkyl,    (C₃-C₈)cycloalkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkyl,    (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl,    aryl, aryl(C₁-C₆)alkyl, Het, Het (C₁-C₆)alkyl, or    (C₁-C₆)alkoxycarbonyl;-   X² is a bond or a linking group; n is 1, 2, 3, or 4; and R³ is a    macromolecule comprising a heterologous peptide, heterologous    protein, heterologous lipid, bead, such as a polystyrene bead,    heterologous nucleic acid molecule or dendrimer;    or a pharmaceutically acceptable salt thereof, including hydrates    thereof.

In certain embodiments, the definition of the group R¹ can be usedinterchangeably with the definition of the group R¹ for any otherformula described herein.

Non-limiting examples of macromolecules or linkers therefore include notonly an oxygen atom, a sulfur atom, a nitrogen atom or a carbon atom(and appropriately appended hydrogen atoms when necessary to fillvalences) but also macromolecules or linkers with side chains thatincrease solubility, such as, for example, groups containing morpholino,piperidino, pyrrolidino, or piperazino rings and the like; amino acids,polymers of amino acids (proteins or peptides), e.g., dipeptides ortripeptides, and the like; carbohydrates (polysaccharides), nucleotidessuch as, for example, PNA, RNA and DNA, and the like; polymers oforganic materials, such as, for example, polyethylene glycol,polylactide and the like; monomeric and polymeric lipids; insolubleorganic nanoparticles; non-toxic body substances such as, for example,cells, lipids, antigens such as, for example microbes, such as, forexample, viruses, bacteria, fungi, and the like. The antigens caninclude inactivated whole organisms, or sub-components thereof and thelike.

Also provided is a method to prevent or inhibit a gram-positivebacterial infection in a mammal. The method includes administering tothe mammal an effective amount of a compound having formula (IB):

-   wherein X¹ is —O—, —S—, or —NR^(c)—;-   wherein R^(c) is hydrogen, C₁₋₁₀alkyl, or substituted C₁₋₁₀alkyl, or    R^(c) and R¹ taken together with the nitrogen atom can form a    heterocyclic ring or a substituted heterocyclic ring, wherein the    substituents on the alkyl, aryl or heterocyclic groups are hydroxy,    C₁₋₆alkyl, hydroxyC₁₋₆alkylene, C₁₋₆alkoxy, C₃₋₆-cycloalkyl,    C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano, halogen, or aryl;-   R¹ is hydrogen, (C₁-C₁₀)alkyl, substituted (C₁-C₁₀)alkyl, C₆₋₁₀aryl,    or substituted C₆₋₁₀aryl, C₅₋₉heterocyclic, substituted    C₅₋₉heterocyclic; wherein the substituents on the alkyl, aryl or    heterocyclic groups are hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkylene,    C₁₋₆alkoxy, C₃₋₆cycloalkyl, C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano,    halogen, or aryl;-   each R² is independently hydrogen, —OH, (C₁-C₆)alkyl, substituted    (C₁-C₆)alkyl, (C₁-C₆)alkoxy, substituted (C₁-C₆)alkoxy,    —C(O)—(C₁-C₆)alkyl(alkanoyl), substituted —C(O)—(C₁-C₆)alkyl,    —C(O)—(C₆-C₁₀)aryl(aroyl), substituted —C(O)—(C₆-C₁₀)aryl, —C(O)OH    (carboxyl), —C(O)O(C₁-C₆)alkyl(alkoxycarbonyl), substituted    —C(O)O(C₁-C₆)alkyl, —NR^(a)R^(b), —C(O)NR^(a)R^(b) (carbamoyl),    substituted —C(O)NR^(a)R^(b), halo, nitro, or cyano; wherein the    substituents on the alkyl, aryl or heterocyclic groups are hydroxy,    C₁₋₆alkyl, hydroxyC₁₋₆alkylene, C₁₋₆alkoxy, C₃₋₆cycloalkyl,    C₁₋₆alkoxyC₁₋₆alkylene, amino, cyano, halogen, or aryl;-   each R^(a) and R^(b) is independently hydrogen, (C₁-C₆)alkyl,    (C₃-C₈)cycloalkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkyl,    (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl,    aryl, aryl(C₁-C₆)alkyl, Het, Het (C₁-C₆)alkyl, or    (C₁-C₆)alkoxycarbonyl;-   X² is a bond or a linking group; n is 1, 2, 3, or 4; and R³ is the    gram-positive bacteria, an isolated antigenic protein or peptide of    the gram-positive bacteria, or an isolated polysaccharide of the    gram-positive bacteria; or a pharmaceutically acceptable salt    thereof, including hydrates thereof. In one embodiment, the    gram-positive bacteria is a Staphylococcus.    In one embodiment, R³ is an isolated antigenic protein or peptide of    Staphylococcus and that compound is administered with a preparation    of killed Staphylococcus.

The invention provides a compound of the invention for use in medicaltherapy (e.g., for prophylaxis of bacterial diseases such as in avaccine). The compounds of the invention can also be used forbiodefense, e., against B. anthrax.

Further provided are compositions and a compound of the invention foruse in medical therapy, e.g., to treat asthma or viral infections orprevent viral infection, as well as the use of the conjugates for themanufacture of a medicament for the treatment of a TLR associatedcondition or symptom or one in which an augmented immune response or asuppressed immune response is indicated

In addition, the invention also provides a pharmaceutical compositioncomprising at least one compound of the invention, or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable diluent or carrier, optionally in combination with apreparation of a selected gram-positive bacteria, e.g., a killedpreparation or an extract, isolated protein of a selected gram-positivebacteria, or isolated carbohydrate (polysaccharide) of a selectedgram-positive bacteria.

The invention includes the use of conjugates of TLR7 agonists and amacromolecule, e.g., one with a primary amino group, that enhances theactivity of the agonist, e.g., albumin, or has a separate desirableactivity, e.g., is an antigen of a gram-positive bacterium. Theconjugates may include macromolecules directly linked to a TLR7 agonist,or linked via a linker to the TLR7 agonist. The conjugates may optimizethe immune response while limiting undesirable systemic side effects ofTLR7 agonists.

In one embodiment, the invention provides a method for preventing ortreating a gram-positive bacterial infection in a mammal, such as ahuman. The method includes administering to a mammal in need of suchtherapy, an effective amount of a compound of the invention, or apharmaceutically acceptable salt thereof, conjugated to an adjuvant orto at least one antigen of the gram-positive bacterium.

Also provided is a method to identify conjugates useful to prevent,inhibit or treat a particular condition or symptom, e.g., by identifyingthe cytokine profile induced by the conjugate in vitro or in vivo or byidentifying the endosome, e.g., early, middle or late, having the TLRfor the TLR agonist. As different cells have different endosomes, theidentification of endosome patterns in cells may allow for targeting ofconjugates to specific cell types or improve access of conjugates toendosomes.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a TLR agonist/alphagalactosyl-ceramide conjugate.

FIG. 2 illustrates a UC-1V150 conjugate of G1 PAMAM with an ethylenediamine core.

FIG. 3A illustrates a linker (SANH) for conjugating a macromolecule anda synthetic TLR agonist.

FIG. 3B illustrates synthesis of UC-1V150.

FIG. 3C illustrates conjugation of UC-1V150 to MSA. 200 μL of MSA (25mg/mL) is mixed with 100 μL of conjugation buffer (1M phosphate, pH=7.2)and 690 μL of PBS. 844 μg of SANH in 10 μL of DMF (40-fold molar excessto MSA) is added to protein solution (Final concentration of NP inreaction mixture is 5 mg/mL). After gentle mixing, reaction is allowedto proceed at room temperature for 2 hours. To remove excess of SANH thereaction mixture is loaded on a microcon spin filter device (YM-3,Millipore) and concentrated to about 70 μL. 460 μg of UC-1V150 dissolvedin 10 μL of DMF was added to MSA modified with SANH and the reactionmixture was incubated at RT overnight. To remove excess UC-1V150 thereaction mixture was first concentrated to 50 μL using a microcon spinfilter device (Millipore: YM-3) and loaded on a G25 micro-spin column(GE Healthcare).

FIG. 4 is a graphic illustration of the absorption profile (at about 350nm) of a compound of formula I (an OVA/SANH/UC-1V150 conjugate).

FIG. 5 illustrates the absorbance profile of a conjugation reaction of asynthetic TLR7 agonist, UC-1V150, to mouse serum albumin (MSA). TheUC-1V150 to MSA ratio is approximately 5:1.

FIG. 6 illustrates a TLR agonist/phospholipid conjugate.

FIG. 7 shows the effect of UC-1V199/lipid administration at differentdoses and timing.

FIGS. 8A-B show that UC-1V199/lipid inhibits TLR7 and TLR2 signaling.

FIG. 9 illustrates biphasic dose-response to an ultrapotent TLR7 agonist(a pico/nanomolar agonist and micromolecular antagonist).

FIGS. 10A and B illustrate that the UC-1V150/MSA conjugates activateboth murine bone marrow-derived macrophages (panel A) and humanperipheral blood mononuclear cells (panel B). Cells were incubated withvarious concentrations of the conjugates from 0.5 nM to 10 μM with BMDMor from 0.1 to 10 μM with PBMC. Culture supernatants were harvestedafter 24 hours and cytokine levels were analyzed by Luminex.

FIGS. 11A, B, and C illustrate the in vivo efficacy of a TLR7 agonistconjugate. C57BL/6 mice were injected (i.v. via the tail vein) withvarious amounts of UC-1V150 (aldehyde-modified SM-360320) orUC-1V150/MSA per mouse. Serum samples were collected and cytokine levelswere analyzed by Luminex. The effect from the unconjugated syntheticTLR7 agonist, SM-360320, lasted for only 2 hours whereas UC-1V150/MSAextended the effect to at least 6 hours.

FIG. 12 shows sustained in vivo local activity of a UC-1V150/MSAconjugate without a systemic effect. C57BL/6 mice were anesthetized andadministered (i.t.) with 3 nmol of UC-1V150/MSA. At the indicated timepoints, mice were sacrificed, and BALF and sera collected. The data werecombined from two separate experiments with at least six mice per group.The results show the mean values±SEM.

FIG. 13 shows cytokine induction in BMDM by irradiated anthraxspore-TLR7 agonist conjugate.

FIGS. 14A-B provide survival graphs after immunization of female A/Jmice (panel A) or Balb/c mice (Panel B) with UC-1V150/MSA and challengewith spores. A) Age matched female A/J mice were administered i.n.saline only or saline containing MSA (an amount equivalent toUC-1V150/MSA), UC-1V150 or UC-1V150/MSA at 0.75 nmole/mouse 1 day beforeB. anthracis infection, and survival was assessed up to 13 days. B)Balb/c mice were administered i.n. saline or UC-1V150/MSA at 5nmole/mouse 1 day before influenza virus infection, and survivalfollowed for 21 days. In each model, Kaplan-Meier survival curves andlog-rank tests were performed to determine significance. At least 8 micewere tested in each group.

FIG. 15 provides a graph of percent survival after a single dose ofvaccine with TLR7 agonists and conjugates.

FIG. 16 illustrates that protection against anthrax spore exposuredepends on CD4+ cells.

FIG. 17 shows a local cytokine profile in mice. C57BL/6 mice wereadministered i.t. with a UC-1V150/MSA conjugate or unconjugated UC-1V136at 3 nmole or 500 nmole per mouse, respectively. BALF and sera werecollected at the indicated time points and cytokine levels determined bymultiplex immunoassay. Mean values from at least 3-5 mice per group areshown ±SEM.

FIG. 18 illustrates the absorbance spectrum for direct conjugation ofSIV particles to UC-1V150.

FIG. 19 illustrates the cytokine induction in BMDC by a conjugate of asynthetic TLR7 agonist and virus particles.

FIGS. 20A-B illustrate the effects of a UC-1V150/inactivated SIVconjugate (panel A) or UC-1V150/OVA/ODN (panel B) on IL-12 production.Myeloid BMDC were incubated for 24 hours under various conditions with0.1 μg/mL as indicated. IL-12 levels in the cell supernatant weremeasured by ELISA.

FIG. 21 is a graphic illustration of the stimulation of bone marrowderived dendritic cells (BMDC) with OVA/UC-1V150 or OVA/ODN(ODN=oligodeoxynucleotide).

FIG. 22 is an illustration of the UV spectrum of a double-conjugate,(OVA/UC-1V150/ODN 1043).

FIG. 23 is an illustration of the induction of IL-12 in BMDC usingOVA/ODN/UC-1V150 conjugates. OVA/1043 and OVA/1018 are ODN conjugates.

FIG. 24 illustrates conjugation of a synthetic TLR agonist to a lipidcomponent of a liposome. Self-assembly of the TLR conjugate coupled viaspacer-linker to a C-15 lipid, resulted in the formation of 100 nMnanoparticles. TLR agonist and an NHS-ester of the lipid were reacted inequimolar amounts in DMF and 1 equivalent of triethylamine for 6 hours.The reaction mixture was purified by preparing HPLC under isocraticconditions in 50:50 acetonitrile/water.

FIG. 25 shows a schematic of a TLR agonist/liposome conjugate. Liposomesare formed with cholesterol:DOPE:DSPC:mPEG2000-DSPE:TLR-DSPE:BODIPY-DOPE30:30:30:5:5:1.5; DSPE=distearoylphosphatidylethanolamine;DOPE=dioleoylphosphatidylethanolamine;BODIPY=6-(((4-4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanamido-DOPE.Cholesterol:DOPE:DSPC:DSPE-TLRagonist:DSPE-mPEG (in 1:1:1:0.16:0.16molar ratio) in chloroform were taken in 30 mL glass culture tubes,dried under a stream of nitrogen gas and vacuum-dessicated for a minimumof 6 h to remove any residual organic solvent. The dried lipid film washydrated in sterile deionized water in a total volume of 1 mL for aminimum of 12 hours. Liposomes were vortexed for 2-3 minutes to removeany adhering lipid film and sonicated in a bath sonicator (ULTRAsonik28X) for 2-3 minutes at room temperature to produce multilamellarvesicles (MLV). MLVs were then sonicated with a Ti-probe (using aBranson 450 sonifier at 100% duty cycle and 25 W output power) in an icebath for 1-2 minutes to produce small unilamellar vesicles (SUVs) asindicated by the formation of a clear translucent solution. The solutionwas pressure filtered in sequence though 200 and then 100 nm nucleoporepolycarbonate membranes to obtain liposome nanoparticles of 100 nm witha polydispersity factor of less than 0.1.

FIG. 26 shows synthesis of lipid conjugate WW-109. 0.45 mg (1 μmole) ofIV-199 was added to 100 μL of a 10 mM solution of DOPE in chloroform. Tothis solution was added 0.1 mg of triethylamine from a chloroform stock.The mixture was reacted at room temperature for 24 hours and thechloroform was rotavaped. The white solid residue was washed three timesin 60%/methanol/hexane and centrifuged to obtain a white solid. The m/zby Mass spec was 1086 and the compound had a uv max absorption at 268nm. Fatty acid moieties of various chain lengths can be used to preparethe analogous compounds, including C₁₄-C₂₂ carboxylic acids with one,two, three, or four sites of unsaturation, epoxidation, hydroxylation,or a combination thereof, at any feasible locations of the carboxylicacid carbon chain. In one specific embodiment, the fatty acid moietiesare C₁₇ carboxylic acids with a site of unsaturation at C₈-C₉. Inanother specific embodiment, the fatty acid moieties are C₁₈ carboxylicacids with a site of unsaturation at C₉-C₁₀. The carboxylic acidmoieties of each fatty acid moiety can be the same, or they can bedifferent (see, e.g., FIG. 6).

FIG. 27 illustrates a schematic of a silica particle with TLR agonistscovalently bonded thereto.

FIGS. 28A-D provide exemplary compounds for preparing conjugates of theinvention or for use in the methods of the invention. A) Structures forUC-1V136, resiquimod, UC-1X105, and UC-1V187. B) Structures forimiquimod, bropirimine, and UC-1V199. C) Structures for UC-1W236,UC-1X51 and loxoribine. D) Structures for UC-1W247, UC-1X113 andUC-1V186. Other conjugates include TLR agonists coupled to human serumalbumin, e.g., HSA/UC-1V150, or DOPE/UC-1V199. UC-1X-51 increasesTNF-alpha levels three fold (110 ng/mL)

DETAILED DESCRIPTION OF INVENTION Definitions

As used herein, the term “antibody” refers to a protein having one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad of immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively.

The basic immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies may exist as intact immunoglobulins, or as modifications in avariety of forms including, for example, FabFc₂, Fab, Fv, Fd, (FabN)₂,an Fv fragment containing only the light and heavy chain variableregions, a Fab or (Fab)N₂ fragment containing the variable regions andparts of the constant regions, a single-chain antibody, e.g., scFv,CDR-grafted antibodies and the like. The heavy and light chain of a Fvmay be derived from the same antibody or different antibodies therebyproducing a chimeric Fv region. The antibody may be of animal(especially mouse or rat) or human origin or may be chimeric orhumanized. As used herein the term “antibody” includes these variousforms.

A composition is comprised of “substantially all” of a particularcompound, or a particular form a compound (e.g., an isomer) when acomposition comprises at least about 90%, and preferably at least about95%, 99%, and 99.9%, of the particular composition on a weight basis. Acomposition comprises a “mixture” of compounds, or forms of the samecompound, when each compound (e.g., isomer) represents at least about10% of the composition on a weight basis. A purine analog of theinvention, or a conjugate thereof, can be prepared as an acid salt or asa base salt, as well as in free acid or free base forms. In solution,certain of the compounds of the invention may exist as zwitterions,wherein counter ions are provided by the solvent molecules themselves,or from other ions dissolved or suspended in the solvent.

As used herein, the term “isolated” refers to in vitro preparation,isolation and/or purification of a nucleic acid molecule, a peptide orprotein, or other molecule so that it is not associated with in vivosubstances or is present in a form that is different than is found innature. Thus, the term “isolated” when used in relation to a nucleicacid, as in “isolated oligonucleotide” or “isolated polynucleotide”refers to a nucleic acid sequence that is identified and separated fromat least one contaminant with which it is ordinarily associated in itssource. An isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. Hence, with respect to an “isolatednucleic acid molecule”, which includes a polynucleotide of genomic,cDNA, or synthetic origin or some combination thereof, the “isolatednucleic acid molecule” (1) is not associated with all or a portion of apolynucleotide in which the “isolated nucleic acid molecule” is found innature, (2) is operably linked to a polynucleotide which it is notlinked to in nature, or (3) does not occur in nature as part of a largersequence. The isolated nucleic acid molecule may be present insingle-stranded or double-stranded form. When a nucleic acid molecule isto be utilized to express a protein, the nucleic acid contains at aminimum, the sense or coding strand (i.e., the nucleic acid may besingle-stranded), but may contain both the sense and anti-sense strands(i.e., the nucleic acid may be double-stranded).

The term “amino acid” as used herein, comprises the residues of thenatural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His,Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) inD or L form, as well as unnatural amino acids (e.g., phosphoserine,phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, citruline, -methyl-alanine,para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine,and tert-butylglycine). The term also comprises natural and unnaturalamino acids bearing a conventional amino protecting group (e.g., acetylor benzyloxycarbonyl), as well as natural and unnatural amino acidsprotected at the carboxy terminus (e.g., as a (C₁-C₆)alkyl, phenyl orbenzyl ester or amide; or as an -methylbenzyl amide). Other suitableamino and carboxy protecting groups are known to those skilled in theart (see for example, T. W. Greene, Protecting Groups In OrganicSynthesis; Wiley: New York, 1981, and references cited therein). Forinstance, an amino acid can be linked to the remainder of a compound offormula I through the carboxy terminus, the amino terminus, or throughany other convenient point of attachment, such as, for example, throughthe sulfur of cysteine.

The term “toll-like receptor” (TLR) refers to a member of a family ofreceptors that bind to pathogen associated molecular patterns (PAMPs)and facilitate an immune response in a mammal. Ten mammalian TLRs areknown, e.g., TLR1-10.

The term “toll-like receptor agonist” (TLR agonist) refers to a moleculethat binds to a TLR. Synthetic TLR agonists are chemical compounds thatare designed to bind to a TLR and activate the receptor. Exemplarysynthetic TLR agonists provided herein include “TLR-7 agonist”, “TLR8agonist”, “TLR-3 agonist” and “TLR-9 agonist.” TLR agonists includeimiquimod, resiquimod, broprimine and loxoribine.

The term “nucleic acid” as used herein, refers to DNA, RNA,single-stranded, double-stranded, or more highly aggregatedhybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, those providing chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, peptide nucleic acids(PNAs), phosphodiester group modifications (e.g., phosphorothioates,methylphosphonates), 2′-position sugar modifications, 5-positionpyrimidine modifications, 7-position purine modifications, 8-positionpurine modifications, 9-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, methylations, unusualbase-pairing combinations such as the isobases, isocytidine andisoguanidine and the like. Nucleic acids can also include non-naturalbases, such as, for example, nitroindole. Modifications can also include3′ and 5′ modifications such as capping with a BHQ, a fluorophore oranother moiety.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the compounds useful in thepresent invention can be synthesized from the parent compound, whichcontains a basic or acidic moiety, by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with a stoichiometric amount of the appropriatebase or acid in water or in an organic solvent, or in a mixture of thetwo; generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., p. 1418 (1985), the disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

The following definitions are used, unless otherwise described: halo orhalogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl,alkynyl, etc. denote both straight and branched groups; but reference toan individual radical such as “propyl” embraces only the straight chainradical, a branched chain isomer such as “isopropyl” being specificallyreferred to. Aryl denotes a phenyl radical or an ortho-fused bicycliccarbocyclic radical having about nine to ten ring atoms in which atleast one ring is aromatic. Het can be heteroaryl, which encompasses aradical attached via a ring carbon of a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is H, O, (C₁-C₄)alkyl,phenyl or benzyl, as well as a radical of an ortho-fused bicyclicheterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene diradical thereto.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine agonist activity using thestandard tests described herein, or using other similar tests which arewell known in the art. It is also understood by those of skill in theart that the compounds described herein include their various tautomers,which can exist in various states of equilibrium with each other.

“Therapeutically effective amount” is intended to include an amount of acompound useful in the present invention or an amount of the combinationof compounds claimed, e.g., to treat or prevent the disease or disorder,or to treat the symptoms of the disease or disorder, in a host. As usedherein, “treating” or “treat” includes (i) preventing a pathologiccondition from occurring (e.g. prophylaxis); (ii) inhibiting thepathologic condition or arresting its development; (iii) relieving thepathologic condition; and/or diminishing symptoms associated with thepathologic condition.

As used herein, the term “patient” refers to organisms to be treated bythe methods of the present invention. Such organisms include, but arenot limited to, mammals such as humans. In the context of the invention,the term “subject” generally refers to an individual who will receive orwho has received treatment (e.g., administration of a compound of theinvention).

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. Only stable compounds are contemplated bythe present invention.

The TLR Agonists and Conjugates of the Invention and Uses Thereof

In one embodiment, the invention provides a therapeutic method forpreventing or treating a pathological condition or symptom in a mammal,such as a human, wherein the activity of a TLR agonist is implicated andits action is desired. The method includes administering to a mammal inneed of such therapy, an effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof. Non-limitingexamples of pathological conditions or symptoms that are suitable fortreatment include cancers, inflammatory diseases of the gastrointestinaltract, brain, skin, joints, and other tissues, bacterial or viraldiseases, autoimmune diseases, and treating Crohn's Disease. Thecompounds of the invention can also be used to prepare vaccines againstbacteria, viruses, cancer cells, or cancer-specific peptides, or toenhance anti-cancer monoclonal antibodies, as a CNS stimulant, or forbiodefense. The invention thus provides a compound of the invention foruse in medical therapy (e.g., for use as an anti-cancer agent, to treatbacterial diseases, to treat for viral diseases, such as hepatitis C andhepatitis B, to treat Crohn's Disease, and generally as therapeuticagents for treating immunologic disease). Furthermore, compounds of theinvention may prevent carcinogenesis, e.g., by hepatitis C and hepatitisB viruses, and may be used for the manufacture of a medicament usefulfor the treatment of cancer, viral or bacterial infection, Crohn'sDisease, and immunologic disorders in a mammal, such as a human.

In one embodiment, the present invention provides a method for treatinga viral infection in a mammal by administering a TLR agonist conjugateof the invention. The viral infection may be caused by a RNA virus, aproduct of the RNA virus that acts as a TLR agonist, and/or a DNA virus.An exemplary DNA virus is hepatitis B virus. In one embodiment, theviral infection is caused by a coronavirus that causes Severe AcuteRespiratory Syndrome (SARS), a Hepatitis B virus, or a Hepatitis CVirus.

In one embodiment, the present invention provides a method for treatingcancer by administering an effective amount of a TLR agonist conjugateof the invention. The cancer may be an interferon sensitive cancer, suchas, for example, a leukemia, a lymphoma, a myeloma, a melanoma, or arenal cancer. Specific cancers that can be treated include melanoma,superficial bladder cancer, actinic keratoses, intraepithelialneoplasia, and basal cell skin carcinoma, squamous, and the like. Inaddition, the method of the invention includes treatment for aprecancerous condition such as, for example, actinic keratoses orintraepithelial neoplasia, familial polyposis (polyps), cervicaldysplasia, cervical cancers, superficial bladder cancer, and any othercancers associated with infection (e.g., lymphoma Karposi's sarcoma, orleukemia); and the like.

In another embodiment, the present invention provides a method fortreating an autoimmune disease by administering a therapeuticallyeffective amount of a TLR agonist conjugate of the invention or apharmaceutically acceptable salt of such a compound. Exemplaryautoimmune diseases are Multiple Sclerosis, lupus, rheumatoid arthritisand the like.

In another embodiment, the present invention provides a method oftreating Crohn's Disease by administering a TLR agonist conjugate of theinvention.

The TLR agonist conjugates may include a homofunctional TLR agonistpolymer, e.g., formed of a TLR7 agonist or a TLR3 agonist. The TLR7agonist can be a 7-thia-8-oxoguanosinyl (TOG) moiety, a7-deazaguanosinyl (7DG) moiety, a resiquimod moiety, or an imiquimodmoiety. In another embodiment, the TLR agonist conjugate may include aheterofunctional TLR agonist polymer. The heterofunctional TLR agonistpolymer may include a TLR7 agonist and a TLR3 agonist or a TLR9 agonist,or all three agonists. The heterofunctional TLR agonist polymer caninclude a TLR8 agonist and a TLR9 agonist.

The invention includes covalently conjugating a synthetic TLR agonistwith selected macromolecules to achieve, for example, a desiredmolecular shape, size and valence in order to optimize the immunologicproperties of the resulting conjugate, and/or to target or deliver theconjugate to desired cells and tissues. As described herein, theconjugates are designed to be useful in a variety of medicalapplications including, but not limited to, allergic asthma, respiratoryviral infections (influenza and RSV), lupus and other autoimmunediseases, and as antigen-adjuvant combinations for vaccines againstcancer and infectious diseases. The conjugates provide an optimum immuneresponse while limiting undesirable systemic side effects by tetheringthe immune activator (the synthetic TLR agonist) to a macromolecule by astrong covalent bond. The macromolecule may serve as a targeting entityand/or an integral part of the immune response, such as the antigen inan adjuvant-antigen conjugate. A major advantage when administering astable conjugate in a localized environment is that only very smallamounts of TLR agonist are released over time into the systemicenvironment.

In one embodiment, the macromolecule is selected from products, such asproteins, lipids or dendrimers, or polymers having amino groups on theirsurfaces, such as polystyrene “amino beads,” each having primary aminogroups available for conjugation to a linker such as SANH, or for directconjugation to the synthetic TLR7 agonist. For example, followingconjugation of the linker and macromolecule, the TLR7 agonist, such asUC-1V150, is reacted with the NHS ester of the SANH-macromoleculeconjugate to provide a TLR7 agonist-SANH-macromolecule conjugate.

Vaccines are not generally used in acute settings because (1) they taketo long to act and (2) they are not effective in immune compromisedpatients. For example, Staphylococcus aureus (SA) infections are a majorcause of morbidity and mortality in hospitalized patients. Groupsparticularly at risk are those with immune suppression due to burns,trauma, catheter placement, dialysis, or advanced age in nursing homes.Moreover, many strains of hospital acquired SA are resistant toconventional antibiotics.

The present invention overcomes these two barriers to treatment. The useof compositions, including synthetic TLR7 agonists and conjugates withsynthetic TLR7 agonists, in combination with gram-positive bacterialantigens is provided herein. TLR7 ligands generally have poorpharmacokinetics, and rapid systemic absorption and excretion. Due tosystemic dispersal, they result in cytokine syndrome. Effectiveadjuvants must create an “immune gradient” of cytokines and chemokines.In one embodiment, conjugation of potent synthetic TLR7 agonists tomacromolecules enhances delivery properties, improves pharmacokinetics,and avoids systemic toxicity by localized exposure.

In one embodiment, the invention provides a method to prevent or inhibita gram-positive bacterial infection in a mammal, comprisingadministering to the mammal an effective amount of a compositioncomprising a bacterial antigen of a gram-positive bacteria and an amountof a synthetic TLR7 agonist. In another embodiment, the inventionprovides a method to prevent or inhibit a gram-positive bacterialinfection in a mammal, comprising administering to the mammal aneffective amount of a synthetic TLR7 agonist conjugated to agram-positive bacterial antigen. For example, a 1V150-MSA conjugateretains its TLR7 agonist activity, has enhanced potency and reducedtoxicity, causes local activation of innate immunity, and induces T celldependent immune protection within 6 days after a single vaccinationwith a bacterial antigen.

In one embodiment, a synthetic TLR7 agonist is administered with orconjugated to one or more antigens of S. aureus. Table 1 providesexemplary antigens for S. aureus for use with synthetic TLR7 agonists,particularly in acute care settings. The vaccines of the invention mayunexpectedly provide a rapid and effective immune response.

TABLE 1 Staphylococcus aureus immunogens Weapon Exfoliative toxin BExfoliative toxin A Toxic shock-syndrome toxin Enterotoxin A-E, H-U Bonesialoprotein-binding protein Collagen-binding protein Clumping factor AClumping factor B α-hemolysin γ-hemolysin Protein A Clumping factor AFibronectin-binding protein A Fibronectin-binding protein BCollagen-binding protein Lipoteichoic acid Peptidoglycan Protein AFibronectin-binding protein B α-hemolysin Panton valentine leukocidinCollagen-binding protein Lipoteichoic acid Peptidoglycan Capsularpolysaccharide Clumping factor A Protein A Fibronectin-binding proteins

In one embodiment, the invention provides the following conjugates

X¹=—O—, —S—, or —NR^(c)—,

wherein R^(c) hydrogen, C₁₋₁₀alkyl, or C₁₋₁₀alkyl substituted by C₃₋₆cycloalkyl, or R^(c) and R¹ taken together with the nitrogen atom canform a heterocyclic ring or a substituted heterocyclic ring, wherein thesubstituents are hydroxy, C₁₋₆ alkyl, hydroxy C₁₋₆ alkylene, C₁₋₆alkoxy, C₁₋₆ alkoxy C₁₋₆ alkylene, or cyano;

wherein R¹ is (C₁-C₁₀)alkyl, substituted (C₁-C₁₀)alkyl, C₆₋₁₀ aryl, orsubstituted C₆₋₁₀ aryl, C₅₋₉ heterocyclic, substituted C₅₋₉heterocyclic; wherein the substituents on the alkyl, aryl orheterocyclic groups are hydroxy, C₁₋₆ alkyl, hydroxy C₁₋₆ alkylene, C₁₋₆alkoxy, C₁₋₆ alkoxy C₁₋₆ alkylene, amino, cyano, halogen, or aryl;

each R² is independently hydrogen, —OH, (C₁-C₆)alkyl, substituted(C₁-C₆)alkyl, (C₁-C₆)alkoxy, substituted (C₁-C₆)alkoxy,—C(O)—(C₁-C₆)alkyl(alkanoyl), substituted —C(O)—(C₁-C₆)alkyl,—C(O)—(C₆-C₁₀)aryl(aroyl), substituted —C(O)—(C₆-C₁₀)aryl, —C(O)OH(carboxyl), —C(O)O(C₁-C₆)alkyl(alkoxycarbonyl), substituted—C(O)O(C₁-C₆)alkyl, —NR^(a)R^(b), —C(O)NR^(a)R^(b) (carbamoyl),—O—C(O)NR^(a)R^(b), —(C₁-C₆)alkylene-NR^(a)R^(b),—(C₁-C₆)alkylene-C(O)NR^(a)R^(b), halo, nitro, or cyano;

wherein each R^(a) and R^(b) is independently hydrogen, (C₁₋₆)alkyl,(C₃-C₈)cycloalky, (C₁₋₆6)alkoxy, halo(C₁₋₆)alkyl,(C₃-C₈)cycloalkyl(C₁₋₆)alkyl, (C₁₋₆)alkanoyl, hydroxy(C₁₋₆)alkyl, aryl,aryl(C₁₋₆)alkyl, aryl, aryl(C₁₋₆)alkyl, Het, Het (C₁₋₆)alkyl, or(C₁₋₆)alkoxycarbonyl; wherein X² is a bond or a linking group; whereinR³ is a macromolecule; wherein n is 1, 2, 3, or 4; wherein m is 1 or 2;wherein q is 1 to 1,000, 10⁴, 10⁵, 10⁶ or more; or a pharmaceuticallyacceptable salt thereof. The macromolecule groups can include organicmolecules, composed of carbon, oxygen, hydrogen, nitrogen, sulfur,phosphorous, or combinations thereof, which are not harmful to bodytissues (e.g., they are non-toxic, and/or do not cause inflammation) andmay include, but not be limited to, dendrimers, proteins, peptides,lipids and their formulations (e.g., liposome nanoparticles), with orwithout linkers (X² groups), and amino-modified polymers, such aspolystyrene beads, as well as α-galactosylceramides (see FIG. 1).

The compounds of the invention can be prepared using compounds havingformula (IA-1):

where X² is a group that can react to a specific group of compounds,e.g., those disclosed in U.S. Pat. No. 6,329,381 (Kurimoto et al.), orform a bond to a linking group or react to form a bond to amacromolecule, and the remaining variables are as defined above forformula (IA). Non-limiting examples of macromolecules include those withside chains that increase solubility, such as, for example, groupscontaining morpholino, piperidino, pyrrolidino, or piperazino rings andthe like; amino acids, polymers of amino acids (proteins or peptides),e.g., dipeptides or tripeptides, and the like; carbohydrates(polysaccharides), nucleotides such as, for example, PNA, RNA and DNA,and the like; polymers of organic materials, such as, for example,polyethylene glycol, poly-lactide and the like; monomeric and polymericlipids; insoluble organic nanoparticles; non-toxic body substances suchas, for example, cells, lipids, vitamins, co-factors, antigens such as,for example microbes, such as, for example, viruses, bacteria, fungi,and the like. The antigens can include inactivated whole organisms, orsub-components thereof, e.g., cells and the like.

In one embodiment, a compound of the invention has formula (IC):

wherein

X is N or CR^(x) wherein R^(x) is hydrogen, halogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, or unsubstitutedheteroalkyl;

Y is S or N;

the dashes (----) indicate optional bonds; wherein:

when the bond between Y and the carbon marked by an asterisk is a doublebond, Q² is not present;

when the bond between Q¹ and the carbon marked by an asterisk is adouble bond, Q¹ is O, S, NY¹, or NNY²Y³; and

when the bond between Q¹ and the carbon marked by an asterisk is asingle bond, Q¹ is hydrogen, cyano, nitro, O—Y², S—Y², NY′Y², orNY²NY³Y⁴; wherein

Y¹ is hydrogen, substituted alkyl, unsubstituted alkyl, substitutedcycloalkyl, unsubstituted cycloalkyl, substituted heteroalkyl,unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, —C(═O)— substitutedalkyl, —C(═O)— unsubstituted alkyl, —C(═O)O— substituted alkyl, —C(═O)O—unsubstituted alkyl, cyano, nitro, hydroxyl, or O—Y²;

Y², Y³, and Y⁴, are each independently hydrogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl;

Z is O, S, or NY⁵ wherein Y⁵ is hydrogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl;

Q² and Q³ are each independently hydrogen, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl;

X¹ is —O—, —S—, or —NR^(c)—;

R^(c) is hydrogen, C₁₋₁₀alkyl, or substituted C₁₋₁₀alkyl, or R^(c) andR¹ taken together with the nitrogen atom can form a heterocyclic ring ora substituted heterocyclic ring;

R¹ is hydrogen, (C₁-C₁₀)alkyl, substituted (C₁-C₁₀)alkyl, C₆₋₁₀aryl, orsubstituted C₆₋₁₀aryl, C₅₋₉heterocyclic, or substituted C₅₋₉heterocyclicring;

each R² is independently hydrogen, —OH, (C₁-C₆)alkyl, substituted(C₁-C₆)alkyl, (C₁-C₆)alkoxy, substituted (C₁-C₆)alkoxy,—C(O)—(C₁-C₆)alkyl(alkanoyl), substituted —C(O)—(C₁-C₆)alkyl,—C(O)—(C₆-C₁₀)aryl(aroyl), substituted —C(O)—(C₆-C₁₀)aryl, —C(O)OH(carboxyl), —C(O)O(C₁-C₆)alkyl(alkoxycarbonyl), substituted—C(O)O(C₁-C₆)alkyl, —NR^(a)R^(b), —C(O)NR^(a)R^(b) (carbamoyl),—O—C(O)NR^(a)R^(b), —(C₁-C₆)alkylene-NR^(a)R^(b),—(C₁-C₆)alkylene-C(O)NR^(a)R^(b), halo, nitro, or cyano;

each R^(a) and R^(b) is independently hydrogen, (C₁-C₆)alkyl,(C₃-C₈)cycloalkyl, (C₁-C₆)heteroalkyl, (C₁-C₆)alkoxy, halo(C₁-C₆)alkyl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₆)alkanoyl, hydroxy(C₁-C₆)alkyl,aryl, aryl(C₁-C₆)alkyl, Het, Het (C₁-C₆)alkyl, or (C₁-C₆)alkoxycarbonyl;

wherein the substituents on any alkyl, cycloalkyl, heteroalkyl, amino,alkoxy, alkanoyl, aryl, heteroaryl, or heterocyclic groups are one ormore (e.g., 1, 2, 3, 4, 5, or 6) hydroxy, C₁₋₆alkyl,hydroxyC₁₋₆alkylene, C₁₋₆alkoxy, C₃₋₆cycloalkyl, C₁₋₆alkoxyC₁₋₆alkylene,amino, cyano, halogen, heterocycle (such as piperidinyl or morpholinyl),or aryl;

X² is a bond or a linking group;

k is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, or 4; and

R³ is a macromolecule comprising a cell, virus, vitamin, cofactor,peptide, protein, nucleic acid molecule, lipid, bead or particle, suchas a polystyrene bead or nanoparticles, or a dendrimer;

or a pharmaceutically acceptable salt thereof, including hydratesthereof.

In certain embodiments, the groups X²—R³ can form a linker to a secondformula (IC) moiety so as to form a dimer. For example, the linker canbe any linker as described herein, such as a divalent aryl orheteroaryl, bis-amide aryl, bis-amide heteroaryl, bis-hydrazide aryl,bis-hydrazide heteroaryl, or the like. Alternatively, Q¹ can form alinker to a second formula (IC) moiety so as to form a dimer through adisulfide linkage. See for example, FIG. 28.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently basic compound such asan amine with a suitable acid affording a physiologically acceptableanion. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts of carboxylic acids canalso be made.

Alkyl includes straight or branched C₁₋₁₀ alkyl groups, e.g., methyl,ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, 1-methylpropyl,3-methylbutyl, hexyl, and the like.

Lower alkyl includes straight or branched C₁₋₆ alkyl groups, e.g.,methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl,2,2-dimethylpropyl, and the like.

The term “alkylene” refers to a divalent straight or branchedhydrocarbon chain (e.g., ethylene: —CH₂—CH₂—).

C₃₋₇ Cycloalkyl includes groups such as, cyclopropyl, cyclopentyl,cyclohexyl, cycloheptyl, and the like, and alkyl-substituted C₃₋₇cycloalkyl group, preferably straight or branched C₁₋₆ alkyl group suchas methyl, ethyl, propyl, butyl or pentyl, and C₅₋₇ cycloalkyl groupsuch as, cyclopentyl or cyclohexyl, and the like.

Lower alkoxy includes C₁₋₆ alkoxy groups, such as methoxy, ethoxy orpropoxy, and the like.

Lower alkanoyl includes C₁₋₆ alkanoyl groups, such as formyl, acetyl,propanoyl, butanoyl, pentanoyl or hexanoyl, and the like.

C₇₋₁₁ aroyl, includes groups such as benzoyl or naphthoyl;

Lower alkoxycarbonyl includes C₂₋₇ alkoxycarbonyl groups, such asmethoxycarbonyl, ethoxycarbonyl or propoxycarbonyl, and the like.

Lower alkylamino group means amino group substituted by C₁₋₆ alkylgroup, such as, methylamino, ethylamino, propylamino, butylamino, andthe like.

Di(lower alkyl)amino group means amino group substituted by the same ordifferent and C₁₋₆ alkyl group (e.g., dimethylamino, diethylamino,ethylmethylamino).

Lower alkylcarbamoyl group means carbamoyl group substituted by C₁₋₆alkyl group (e.g., methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl,butylcarbamoyl).

Di(lower alkyl)carbamoyl group means carbamoyl group substituted by thesame or different and C₁₋₆ alkyl group (e.g., dimethylcarbamoyl,diethylcarbamoyl, ethylmethylcarbamoyl).

Halogen atom means halogen atom such as fluorine atom, chlorine atom,bromine atom or iodine atom.

Aryl refers to a C₆₋₁₀ monocyclic or fused cyclic aryl group, such asphenyl, indenyl, or naphthyl, and the like.

Heterocyclic or heterocycle refers to monocyclic saturated heterocyclicgroups, or unsaturated monocyclic or fused heterocyclic group containingat least one heteroatom, e.g., 0-3 nitrogen atoms (—NR^(d)— where R^(d)is H, alkyl, or Y² as defined herein), 0-1 oxygen atom (—O—), and 0-1sulfur atom (—S—). Non-limiting examples of saturated monocyclicheterocyclic group includes 5 or 6 membered saturated heterocyclicgroup, such as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl,piperazinyl or pyrazolidinyl. Non-limiting examples of unsaturatedmonocyclic heterocyclic group includes 5 or 6 membered unsaturatedheterocyclic group, such as furyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, thienyl, pyridyl or pyrimidinyl. Non-limiting examples ofunsaturated fused heterocyclic groups includes unsaturated bicyclicheterocyclic group, such as indolyl, isoindolyl, quinolyl,benzothizolyl, chromanyl, benzofuranyl, and the like. A Het group can bea saturated heterocyclic group or an unsaturated heterocyclic group,such as a heteroaryl group.

R^(c) and R¹ taken together with the nitrogen atom to which they areattached can form a heterocyclic ring. Non-limiting examples ofheterocyclic rings include 5 or 6 membered saturated heterocyclic rings,such as 1-pyrrolidinyl, 4-morpholinyl, 1-piperidyl, 1-piperazinyl or1-pyrazolidinyl, 5 or 6 membered unsaturated heterocyclic rings such as1-imidazolyl, and the like.

The alkyl, aryl, heterocyclic groups of R¹ can be optionally substitutedwith one or more substituents, wherein the substituents are the same ordifferent, and include lower alkyl; cycloalkyl, hydroxyl; hydroxy C₁₋₆alkylene, such as hydroxymethyl, 2-hydroxyethyl or 3-hydroxypropyl;lower alkoxy; C₁₋₆ alkoxy C₁₋₆ alkyl, such as 2-methoxyethyl,2-ethoxyethyl or 3-methoxypropyl; amino; alkylamino; dialkyl amino;cyano; nitro; acyl; carboxyl; lower alkoxycarbonyl; halogen; mercapto;C₁₋₆ alkylthio, such as, methylthio, ethylthio, propylthio or butylthio;substituted C₁₋₆ alkylthio, such as methoxyethylthio,methylthioethylthio, hydroxyethylthio or chloroethylthio; aryl;substituted C₆₋₁₀ monocyclic or fused-cyclic aryl, such as4-hydroxyphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl or3,4-dichlorophenyl; 5-6 membered unsaturated heterocyclic, such asfuryl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl orpyrimidinyl; and bicyclic unsaturated heterocyclic, such as indolyl,isoindolyl, quinolyl, benzothiazolyl, chromanyl, benzofuranyl orphthalimino. In certain embodiments, one or more of the above groups canbe expressly excluded as a substituent of various other groups of theformulas.

In some embodiments, the five-membered ring of the formula is a thiazolering, e.g., where Y of formula IA above is S and Q² is absent.

The alkyl, aryl, heterocyclic groups of R² can be optionally substitutedwith one or more substituents, wherein the substituents are the same ordifferent, and include hydroxyl; C₁₋₆ alkoxy, such as methoxy, ethoxy orpropoxy; carboxyl; C₂₋₇ alkoxycarbonyl, such as methoxycarbonyl,ethoxycarbonyl or propoxycarbonyl) and halogen.

The alkyl, aryl, heterocyclic groups of R^(c) can be optionallysubstituted with one or more substituents, wherein the substituents arethe same or different, and include C₃₋₆ cycloalkyl; hydroxyl; C₁₋₆alkoxy; amino; cyano; aryl; substituted aryl, such as 4-hydroxyphenyl,4-methoxyphenyl, 4-chlorophenyl or 3,4-dichlorophenyl; nitro andhalogen.

The heterocyclic ring formed together with R^(c) and R¹ and the nitrogenatom to which they are attached can be optionally substituted with oneor more substituents, wherein the substituents are the same ordifferent, and include C₁₋₆ alkyl; hydroxy C₁₋₆ alkylene; C₁₋₆ alkoxyC₁₋₆ alkylene; hydroxyl; C₁₋₆ alkoxy; and cyano.

In some embodiments, when Q¹ is O—Y², Y² is not hydrogen.

A specific value for X is N.

Another specific value for X is CH.

Of course, only one of the two bonds indicated by dashed lines may bepresent in one molecule of a compound of the indicated formula. In oneembodiment, the bond between Y and the carbon marked by an asterisk is adouble bond. In another embodiment, the bond between Q¹ and the carbonmarked by an asterisk is a double bond.

A specific value for Q¹ is O.

Another specific value for Q¹ is S.

Another specific value for Q¹ is NY¹, for example, ═NH.

Another specific value for Q¹ is NNY²Y³.

In one embodiment, the bond between Q¹ and the carbon marked by anasterisk is a single bond.

A specific value for Q¹ is hydrogen.

Another specific value for Q¹ is NH₂.

Another specific value for Q¹ is O—Y².

A specific value for Y¹ is hydrogen.

Another specific value for Y¹ is alkyl, for example, (C₁-C₆)alkyl, suchas methyl.

Another specific value for Y¹ is aryl, such as phenyl.

A specific value for each of Y², Y³, and Y⁴ is hydrogen.

Another specific value for each of Y², Y³, and Y⁴ (independently) isalkyl, for example, (C₁-C₆)alkyl, such as methyl.

Another specific value for each of Y², Y³, and Y⁴ (independently) isaryl, such as phenyl.

A specific value for Z is O.

Another specific value for Z is S.

Another specific value for Z is NY⁵ wherein Y⁵ is hydrogen, methyl, orphenyl.

A specific value for Q² is hydrogen.

Another specific value for Q² is methyl, or phenyl.

A specific value for Q³ is hydrogen.

Another specific value for Q³ is methyl, or phenyl.

A specific value for X¹ is a sulfur atom, an oxygen atom or —NR^(c)—.

Another specific X¹ is a sulfur atom.

Another specific X¹ is an oxygen atom.

Another specific X¹ is —NR^(c)—.

Another specific X¹ is —NH—.

A specific value for Y is N.

Another specific value for Y is S.

A specific value for R^(c) is hydrogen, C₁₋₄ alkyl or substituted C₁₋₄alkyl.

A specific value for R¹ and R^(c) taken together is when they form aheterocyclic ring or a substituted heterocyclic ring.

Another specific value for R¹ and R^(c) taken together is substituted orunsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring

A specific value for R¹ is hydrogen, C₁₋₄alkyl, or substitutedC₁₋₄alkyl.

Another specific R¹ is 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl,2-aminoethyl, 3-aminopropyl, 4-aminobutyl, methoxymethyl,2-methoxyethyl, 3-methoxypropyl, ethoxymethyl, 2-ethoxyethyl,methylthiomethyl, 2-methylthioethyl, 3-methylthiopropyl, 2-fluoroethyl,3-fluoropropyl, 2,2,2-trifluoroethyl, cyanomethyl, 2-cyanoethyl,3-cyanopropyl, methoxycarbonylmethyl, 2-methoxycarbonylethyl,3-methoxycarbonylpropyl, benzyl, phenethyl, 4-pyridylmethyl,cyclohexylmethyl, 2-thienylmethyl, 4-methoxyphenylmethyl,4-hydroxyphenylmethyl, 4-fluorophenylmethyl, or 4-chlorophenylmethyl.

Another specific R¹ is hydrogen, CH₃—, CH₃—CH₂—, CH₃CH₂CH₂—,hydroxyC₁₋₄alkylene, or C₁₋₄alkoxyC₁₋₄alkylene.

Another specific value for R¹ is hydrogen, CH₃—, CH₃—CH₂—, CH₃—O—CH₂CH₂—or CH₃—CH₂—O—CH₂CH₂—.

A specific value for R² is hydrogen, halogen, or C₁₋₄alkyl.

Another specific value for R² is hydrogen, chloro, bromo, CH₃—, orCH₃—CH₂—.

Specific substituents for substitution on the alkyl, aryl orheterocyclic groups are hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkylene,C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkylene, C₃₋₆cycloalkyl, amino, cyano,halogen, or aryl.

A specific value for X² is a bond or a chain having up to about 24atoms; wherein the atoms are selected from the group consisting ofcarbon, nitrogen, sulfur, non-peroxide oxygen, and phosphorous.

Another specific value for X² is a bond or a chain having from about 4to about 12 atoms.

Another specific value for X² is a bond or a chain having from about 6to about 9 atoms.

Another specific value for X² is

Another specific value for X² is

In certain embodiments, the linker or the group X² is not a linkerdisclosed in PCT Application Publication No. WO 2007/024707.Additionally, in some embodiments, R³ is not an auxiliary disclosed inPCT Application Publication No. WO 2007/024707.

A specific macromolecule is an amino acid, a carbohydrate, a peptide, aprotein, an antigen, a nucleic acid, a lipid, a dendrimer, a bodysubstance, or a cell such as a microbe.

A specific peptide, has from 2 to about 20 amino acid residues.

Another specific peptide, has from 10 to about 20 amino acid residues.

A specific macromolecule includes a carbohydrate.

A specific nucleic acid is DNA, RNA or PNA.

A specific macromolecule is a cell, lipid, vitamin, lipid, or co-factor.

A specific antigen is a microbe.

A specific microbe is a virus, bacteria, or fungi.

Another specific microbe is a virus or a bacteria.

Specific bacteria are Bacillus anthracis, Listeria monocytogenes,Francisella tularensis, Salmonella, or Staphylococcus.

Specific Salmonella are S. typhimurium or S. enteritidis.

Specific Staphylococcus include S. aureus.

Specific viruses are RNA viruses, including RSV and influenza virus, aproduct of the RNA virus, or a DNA virus, including herpes virus.

A specific DNA virus is hepatitis B virus.

In other embodiments, the macromolecule is not an amino acid, acarbohydrate, a peptide, an antigen such as a microbe, for example, avirus (for example, RNA viruses, e.g., SIV, hepatitis C virus or acoronavirus, a product of the RNA virus, or a DNA virus, such asHepatitis B virus, fungi, or bacteria such as Bacillus anthracis(anthrax), Listeria monocytogenes, Francisella tularensis, or Salmonella(e.g., typhimurium or enteritidis), a nucleic acid such as DNA, RNA,PNA, or a body substance such as a cell or lipid.

A specific value for k is 0. Another specific value for k is 1. Anotherspecific value for k is 2. In some embodiments, k is not 1.

Specific compounds of the invention have the general formulaIA-L-A¹;IA-L-(A¹)₂;IA-L-A¹-A¹;IA-L-A¹-L-A¹;(IA)₂-L-A¹-A¹;(IA)₂-L-A¹-L-A¹;(IA)₂-L-A¹; or(IA)₂-L-(A¹)₂;

wherein IA is as disclosed herein; L is absent or is a linking group;and each A¹ group independently represents a macromolecule.

The invention includes compositions of a compound of the inventionoptionally in combination with other active agents, e.g., ribavirin,mizoribine, and mycophenolate mofetil. Other non-limiting examples areknown and are disclosed in U.S. published patent application No.20050004144.

Processes for preparing compounds of the invention for preparingintermediates useful for preparing compounds of the invention areprovided as further embodiments of the invention. Intermediates usefulfor preparing compounds of the invention are also provided as furtherembodiments of the invention.

For example, the compounds (conjugates) of the invention can be preparedusing standard synthetic methods known in the art. A general ester andaldehyde synthesis is illustrated below. UC-1V150 was synthesized inseven steps from 2,6-dichloropurine. The free aldehyde group on thebenzyl moiety of UC-1V150 enabled us to couple the agonist to manydifferent auxiliary chemical entities, including proteins,oligonucleotides, aromatic molecules, lipids, viruses, and cells,through a linker molecule that contained a hydrazine or amino group.

General Synthesis

NHS Ester Synthesis

Aldehyde Synthesis

Chemistry of UC-1V150.

The synthesis of UC-1V150 and the preparation of the indicated compounds2-8 was as follows. Compound 2:4-(2,6-dichloropurin-9-ylmethyl)benzonitrile. 2,6-dichloro-9H-purine (1,16 mmol) was dissolved in DMF (50 mL) with potassium carbonate (50 mmol)added, and the mixture was stirred at ambient temperature for 16 hoursafter adding α-Bromo-p-tolunitrile (22 mmol). After filtration to removeinsoluble inorganic salts, the filtrate was poured into water (1500 mL)and extracted with ethyl acetate (2×400 mL), dried over magnesiumsulfate and evaporated to yield a residue which was subjected to flashsilica gel chromatography using 1:2:10 ethyl acetate/acetone/hexanes.Yield 3.33 g (69%). UV, NMR and MS were consistent with structureassignment. Compound 3: 4-(6-amino-2-chloropurin-9-ylmethylbenzonitrile.Compound 2 (1.9 g) was placed in a steel reaction vessel and methanolicammonia (80 mL, 7 N) was added. The sealed vessel was heated at 60° C.for 12 hours, cooled in ice and the solid product filtered off. Yield1.09 g. UV, NMR and MS were consistent with assigned structure. Compound4: 4-[6-amino-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile. Thesodium salt of 2-methoxyethanol was first generated by dissolving sodiummetal (81 mg) in 2-methoxyethanol (30 mL) with heat, and then compound 3(1.0 g) dissolved in methoxyethanol was added (300 mL, with heat). Thereaction mixture was heated for 8 hours at 115° C. bath temperature,concentrated in vacuo to near dryness and the residue partitionedbetween ethyl acetate and water. Flash silica gel chromatography of theorganic layer using 5% methanol in dichloromethane gave 763 mg product.NMR was consistent with structure assignment. Compound 5:4-[6-amino-8-bromo-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile.Compound 4 (700 mg) was dissolved in dichloromethane (400 mL) andbromine (7 mL) was added dropwise. The mixture was stirred overnight atroom temperature and extracted first with aqueous sodium thiosulfate (2L of 0.1 M) solution, then with aqueous sodium bicarbonate (500 mL,saturated). The residue from the organic layer was chromatographed onsilica gel using 3% methanol in dichloromethane to yield 460 mg of bromoproduct. NMR, W and MS were consistent with structure assignment.Compound 6:4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile.Sodium methoxide was generated by reaction of sodium metal (81 mg) indry methanol (30 mL) and combined with a solution of compound 5 (700 mg)dissolved in dry dimethoxyethane and the temperature raised to 100° C.After overnight reaction, the mixture was concentrated in vacuo and theresidue was chromatographed on silica using 5% methanol indichloromethane. Yield 120 mg. NMR was consistent with structureassignment. Compound 7:4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde.Compound 6 (100 mg) was dissolved in dry THF (3 mL) and cooled to 0° C.under argon. The reducing agent, lithiumN,N′-(dimethylethylenediamino)aluminum hydride, used to convert thenitrile to the aldehyde function. A 0.5 M solution in dry THF wasprepared and 0.72 mL of such was added to the reaction flask. Themixture was stirred at 0-5° C. for 1 hour, quenched by addition of 3 MHCl, extracted with ethyl acetate followed by dichloromethane, and thenconcentrated in vacuo to yield 85 mg. NMR was consistent with structureassignment. Compound 8:4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde(UC-1V150). Compound 7 (800 mg) was combined with sodium iodide (504 mg)and acetonitrile (40 mL), and chlorotrimethylsilane (0.5 mL) was slowlyadded. The mixture was heated at 70° C. for 3.5 hours, cooled andfiltered. The solid product was washed with water, then ether, to yield406 mg. NMR, UV, and MS were consistent with structure assignment.

Additional examples for preparing specific compounds are includedherein.

As described in the examples herein, a soluble TLR7 agonist capable ofcovalent coupling to primary amines under physiologic conditions wasprepared. The in vitro activity of several compounds andantigen-adjuvant complexes was then tested utilizing bone marrow-derivedmurine or peripheral blood mononuclear cell-derived dendritic cells (DC)to characterize DC maturation and cytokine secretion (e.g., IL-12, IL-6,TGF-beta, and IFN-gamma). Immunocompetent syngeneic C57/B1 mice wereprophylactically vaccinated with intradermal antigen-TLR7 agonistcomplexes and challenged with B16 melanoma tumor cells expressing thecOVA transgene.

The effective concentration (EC₅₀) for each compound generally followeda bell shaped distribution with higher doses being inhibitory. Maximalstimulation occurred between 10 and 1000 nM. Covalently coupled adjuvantmolecules to TLR agonist retained activity but with generally lower EC₅₀values. Coupling UC-1V199 to chicken ovalbumin nearly doubled mediansurvival from 22 to 35 days following subcutaneous tumor challengecompared with chicken ovalbumin alone.

Thus, covalent linkage of a TLR7 agonist to a tumor antigen stimulatedDC cytokine production and protected mice from tumor challenge. The useof a suitable TLR7 agonist which retains its immune stimulatingproperties under physiologic conditions following coupling to amacromolecule, such as an antigen, may be useful in the development ofan in situ vaccine in solid tumor therapy.

Various purines, pyridines, and imidazoquinolines, with molecularweights of 200-400 kD, have been shown to activate TLR7 and compoundsthat were specific TLR7 ligands were 100-1000 fold more powerful thanimiquimod on a molar basis (Lee et al., infra). Because these TLRagonists are structurally very similar to normal component ofnucleotides, they are very unlikely to induce a haptenic immune reactionafter repeated administration.

An adenine based TLR7 pharmacore may need to be covalently tied to an“auxiliary group” (macromolecule) to promote uptake into the endosomesof dendritic cells, where TLR7 is expressed, and to retain the TLRagonist. Accordingly, the TLR7 agonist UC-1V150 was prepared and coupledvia its aldehyde function and a linker to free amino groups on variousproteins, including mouse albumin (MSA) (FIG. 3). The conjugates were100-fold more potent in vitro and in vivo than the uncoupled adenineanalog. Moreover, intrapulmonary administration of the albumin conjugate(UC-1V150/MSA) to mice induced local cytokine production in thebronchial alveolar lavage fluid (BALF) without systemic cytokinerelease. In marked contrast, the delivery of the untethered drug to theairways quickly triggered cytokine release in the bloodstream.

In one embodiment, a TLR7 agonist maximizes the production of Th1stimulating cytokines (interferons and IL-12) compared to TNFα and IL-1.TLR7 is localized on the inner surfaces of the endosomal vesicles thatare constantly synthesized and undergo maturation in DC. For example, toprevent asthma, a stable and potent TLR agonist that traffics to theearly endosomes of dendritic cells and induces primarily Type Iinterferons is preferred. A TLR agonist was covalently attached to aphospholipid auxiliary group with the expectation that the conjugate,UC-1V199/L (FIG. 6), would quickly and stably insert into lipidmembranes of cells, including endosomal vesicles. Remarkably, as littleas 30 picomolar UC-1V199/L induced cytokine synthesis in bone marrowderived mouse mononuclear cells. FIGS. 7-8 show data for IL-12synthesis.

The TLR7 ligands that are purines or imidazoquilolines have a peculiarproperty, i.e., a biphasic dose response curve. At high concentrations,the drug fails to induce cytokine synthesis. The biphasic effect isobserved in highly purified dendritic cells, and appears to be cellautonomous. However, the remarkable potency of UC-1V199/L enabledre-examination of the phenomenon, using pharmacologically acceptabledrug concentrations (FIG. 9). Maximal cytokine production was observedwith 10 nM UC-1V199/L while higher concentrations induced progressivelyless IL-12 (and TNF) release.

High and sustained concentrations of TLR7 agonists are known to inducerefractoriness to TLR re-stimulation that can last 24 hours or more.Such a complex system of regulation is apparently part of a fail-safemechanism that prevents cells and tissues from self-destruction duringinflammatory responses. Thus, it was of interest to determine ifconcentrations of UC-1V199/L that failed to induce significant cytokinesynthesis could nonetheless induce “TLR tolerance.” Indeed, when bonemarrow derived mononuclear cells were exposed to a non-activatingconcentration UC-1V199/L (1 μM), and then re-stimulated 24 hours laterwith the same compound, with UC-1V150 or with pam3Cys (P3C, a TLR2activator), they displayed a markedly diminished cytokine response. Incontrast, the UC-1V199/L treated cells retained responsiveness toligands of TLR3 and TLR4, which go through the TRIF pathway (results notshown). Preliminary experiments indicated that non-responsiveness wasalso induced in vivo. Thus, daily administration of UC-1V199/L, andrelated drugs, may suppress inflammation induced by MyD88-dependentstimuli, without the systemic side effects associated with TLRactivation.

In one embodiment, the conjugates of the invention may be useful forpreventing, inhibiting or treating asthma. Asthma is characterized byepisodes of intermittent reversible airway constriction, bronchialsmooth muscle hyperplasia and chronic inflammation. Atopic diseasepredisposes to asthma but up to half of the affected patients are notatopic. Other environmental risk factors for asthma include tobaccosmoke and air pollutants. Moreover, disease flares in affected asthmaticpatients may be triggered not only by allergens but also by airwayirritants, temperature changes and infections.

The initial development of an allergic response is partly regulated by abalance between Th1 and Th2 lymphocytes, and their respective cytokines,especially the interferons and IL-4. Vaccination of animals with anallergen in conjunction with a TLR7 or TLR9 agonist preferentiallyexpands allergen-specific Th1 memory cells. Consequently, subsequentimmunization with antigen in conjunction with a Th2 biased adjuvant doesnot readily elicit an IgE response. Mice that were vaccinated withantigen and TLR7 or TLR9 agonists were resistant to experimental asthma.

A different approach is needed for the treatment of asthma with TLRagonists versus the prevention of asthma. In affected patients, theairways and pulmonary tissues are already infiltrated with a diversepopulation of inflammatory cells, including many subsets of lymphocytes,macrophages, dendritic cells, mast cells, eosinophils, and neutrophils.In this situation, TLR agonists can potentially exacerbate disease, byaugmenting the release of inflammatory mediators such as TNF-alpha andIL-1. Indeed, the ability of various microbial agents to activate TLRsmay explain why they trigger asthmatic attacks.

A TLR agonist for the prevention of asthma preferably is confined to thelungs but also maximizes the production of Th1 stimulating cytokines(interferons and IL-12), compared to TNF-alpha and IL-1. Both TLR7 andTLR9 are localized on the inner surfaces of the endosomal vesicles thatare constantly synthesized and undergo maturation in dendritic cells.TLR9 activating oligonucleotides that are aggregated phosphodiesteroligonucleotides stay longer in early endosomal vesicles and thereforeinduce more type I interferons than nonaggregated phosphorothioateoligonucleotides, which go to mature vesicles. The results imply thatthe spatial organization of the TLR agonist governs its trafficking andits pattern of induced cytokine synthesis. To prevent asthma, a stable,potent and molecularly characterized TLR agonist that trafficks to theearly endosomes of dendritic cells and induces primarily Type Iinterferons is preferred.

To study the effect of conjugates of the invention on allergic asthma,airway inflammation is induced by sensitizing mice via subcutaneaousinjection of 20 μg of ovalbumin absorbed with 500 μg alum per mouse insaline on day 0 and day 7. On days 16 and 21, mice are challenged i.n.with 5 μg ovalbumin per mouse. Conjugates are administered i.n., p.o. ori.v. at different time points prior to the first ovalbumin challenge onday 16. Twenty-four hours after the last challenge (day 22), airwayresponsiveness is measured, mice are sacrificed and BALF cells, lung andspleen samples collected. Naïve mice and ovalbumin/alum-sensitized miceserve as controls. The total number of cells in BALF are counted andstained with Wright-Giemsa to determine numbers of eosinophils,lymphocytes, neutrohils and last cells. Cytokine levels in the BALF aredetermined by Luminex assays. Airway responsiveness to methacholine isassessed 24 hours after the last challenge using a single chamber, wholebody plethysmograph. The Penh, a dimensionless value that correlateswell with pulmonary resistance measured by conventional two chamberplethysmography in ventilated mice, is used to monitor airwayresponsiveness.

The compounds of this invention are administered in a therapeuticallyeffective amount to a subject in need of treatment. Administration ofthe compositions of the invention can be via any of suitable route ofadministration, particularly parenterally, for example, intravenously,intra-arterially, intraperitoneally, intrathecally, intraventricularly,intraurethrally, intrasternally, intracranially, intramuscularly, orsubcutaneously. Such administration may be as a single bolus injection,multiple injections, or as a short- or long-duration infusion.Implantable devices (e.g., implantable infusion pumps) may also beemployed for the periodic parenteral delivery over time of equivalent orvarying dosages of the particular formulation. For such parenteraladministration, the compounds are preferably formulated as a sterilesolution in water or another suitable solvent or mixture of solvents.The solution may contain other substances such as salts, sugars(particularly glucose or mannitol), to make the solution isotonic withblood, buffering agents such as acetic, critric, and/or phosphoric acidsand their sodium salts, and preservatives.

The compounds of the invention can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

In addition, in one embodiment, the invention provides various dosageformulations of the conjugates for inhalation delivery. For example,formulations may be designed for aerosol use in devices such asmetered-dose inhalers, dry powder inhalers and nebulizers.

Examples of useful dermatological compositions which can be used todeliver the compounds of the invention to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the invention can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949. The ability of a compound of the invention to act asa TLR agonist may be determined using pharmacological models which arewell known to the art, including the procedures disclosed by Lee et al.,Proc. Natl. Acad. Sci. USA, 100: 6646 (2003).

Generally, the concentration of the compound(s) of the invention in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The active ingredient may be administered to achieve peak plasmaconcentrations of the active compound of from about 0.5 to about 75 μM,preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM.This may be achieved, for example, by the intravenous injection of a0.05 to 5% solution of the active ingredient, optionally in saline, ororally administered as a bolus containing about 1-100 mg of the activeingredient. Desirable blood levels may be maintained by continuousinfusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the active ingredient(s).

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. In general, however, a suitable dose will be in the range offrom about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kgof body weight per day, such as 3 to about 50 mg per kilogram bodyweight of the recipient per day, preferably in the range of 6 to 90mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye. The dose, and perhapsthe dose frequency, will also vary according to the age, body weight,condition, and response of the individual patient. In general, the totaldaily dose range for a compound or compounds of formula (I), for theconditions described herein, may be from about 50 mg to about 5000 mg,in single or divided doses. Preferably, a daily dose range should beabout 100 mg to about 4000 mg, most preferably about 1000-3000 mg, insingle or divided doses, e.g., 750 mg every 6 hr of orally administeredcompound. This can achieve plasma levels of about 500-750 uM, which canbe effective to kill cancer cells. In managing the patient, the therapyshould be initiated at a lower dose and increased depending on thepatient's global response.

As described above, compositions that contain a compound of theinvention, are useful in the treatment or prevention of a disease ordisorder in, for example, humans or other mammals (e.g., bovine, canine,equine, feline, ovine, and porcine animals), and perhaps other animalsas well. Depending on the particular compound, the composition will, forexample, be useful for treating cancer, an infection, enhancing adaptiveimmunity (e.g., antibody production, T cell activation, etc.), asvaccines, and/or stimulating the central nervous system.

The invention will be further described by the following non-limitingexamples.

Example 1

Processes for preparing compounds of formula (I) are provided as furtherembodiments of the invention and are illustrated by the followingprocedures in which the meanings of the generic radicals are as givenabove unless otherwise qualified.

General Chemistry.

Reagents and solvents were acquired from Aldrich, Milwaukee, Wis.Uncorrected melting points were determined on a Laboratory DeviceMel-Temp II capillary melting point apparatus. Proton nuclear magneticresonance spectra were recorded on a Varian Unity 500 NMRspectrophotometer at 499.8 MHz or on a Varian Mercury NMRspectrophotometer at 400.06 MHz. The chemical shifts were reported inppm on the scale from the indicated reference. Positive and negative ionloop mass spectra were performed by Department of Chemistry UCSD, SanDiego, Calif. Elemental analyses were performed by NuMega ResonanceLabs, San Diego, Calif. Column chromatography was conducted on E Mercksilica gel (230-400 mesh) with the indicated solvent system. Analyticalthin layer chromatography (TLC) was conducted on silica gel 60 F-254plates (EM Reagents).

Preparation of 4-(2,6-dichloropurin-9-ylmethyl)benzonitrile

2,6-dichloro-9H-purine (16 mmol) is dissolved in DMF (50 mL) andpotassium carbonate (50 mmol) is added. α-Bromo-p-tolunitrile (22 mmol)is then added and the mixture is stirred at ambient temperature for 16hours. After filtration to remove insoluble inorganic salts, thefiltrate is poured into water (1500 mL) and extracted with ethyl acetate(2×400 mL), dried over magnesium sulfate and evaporated to yield aresidue which is subjected to flash silica gel chromatography using1:2:10 ethyl acetate/acetone/hexanes. Yield 3.33 g (69%). UV, NMR and MSwere consistent with structure assignment.

Preparation of 4-(6-amino-2-chloropurin-9-ylmethylbenzonitrile

The product above (1.9 g) is placed in a steel reaction vessel andmethanolic ammonia (80 mL, 7 N) is added. The sealed vessel is heated at60° C. for 12 hours, cooled in ice and the solid product filtered off.Yield 1.09 g. UV, NMR and MS were consistent with assigned structure.

Preparation of4-[6-amino-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile

Sodium salt of 2-methoxyethanol is generated by dissolving sodium metal(81 mg) in 2-methoxyethanol (30 mL) with heat. To this solution is addedthe product of example 2 (1.0 g) dissolved in methoxyethanol (300 mL,with heat). The reaction mixture is heated for 8 hours at 115° C. bathtemperature, concentrated in vacuo to near dryness and the residuepartitioned between ethyl acetate and water. Flash silica gelchromatography of the organic layer using 5% methanol in dichloromethanegave 763 mg product. NMR is consistent with structure assignment.

Preparation of4-[6-amino-8-bromo-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile

The product immediately above (700 mg) is dissolved in dichloromethane(400 mL) and bromine (7 mL) is added dropwise. The mixture is stirredovernight at room temperature and extracted with aqueous sodiumthiosulfate (2 L of 0.1 M) solution and then with aqueous sodiumbicarbonate (500 mL, saturated). The residue from the organic layer ischromatographed on silica gel using 3% methanol in dichloromethane) toyield 460 mg of bromo product. NMR, UV and MS are consistent withstructure assignment.

Preparation of4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile

Sodium methoxide is generated by reaction of sodium metal (81 mg) in drymethanol (30 mL). The product immediately above (700 mg) is dissolved indry dimethoxyethane and the temperature raised to 100° C. Afterovernight reaction, the mixture is concentrated in vacuo and the residueis chromatographed on silica using 5% methanol in dichloromethane. Yield120 mg. NMR is consistent with structure assignment.

Preparation of Lithium N,N′-(dimethylethylenediamino)aluminum hydride

This reducing agent used to convert the nitrile to the aldehyde functionis prepared essentially as described in Bull. Korean Chem. Soc., 23:1697(2002). A 0.5 M solution in dry THF is prepared.

Preparation of4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde

4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile(100 mg) is dissolved in dry THF (3 mL) and cooled to 0° C. under argon.The aluminum hydride reagent generated above (0.72 mL) is added to thereaction flask and the mixture is stirred at 0-5° C. for 1 hour and thenquenched by addition of 3 M HCl. The mixture is then extracted withethyl acetate and then dichloromethane and concentrated in vacuo toyield 85 mg. NMR is consistent with structure assignment.

Preparation of4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde(UC-1V150)

The product immediately above (800 mg) is combined with sodium iodide(504 mg) and acetonitrile (40 mL), and then chlorotrimethylsilane (0.5mL) is slowly added. The mixture is heated at 70° C. for 3.5 hours,cooled and filtered. The solid product is washed with water, then etherto yield 406 mg. NMR, UV, MS are consistent with structure assignment.This material is suitable for conjugation reactions between linkers andmacromolecules.

Preparation of methyl4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate

The procedure is as described by Jayachitra, et al., Synth. Comm.,33:3461 (2003)).4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzonitrile (1mmol) is dissolved in dry methanol (5 mL) and freshly distilled BF₃etherate (4 mmol) is added to the solution. The resulting mixture isrefluxed under argon for 20 hours. The solvent is removed in vacuo andthe residue is taken up in dichloromethane (10 mL) and extracted withdilute aqueous sodium bicarbonate (2×10 mL) and the organic layer isdried over magnesium sulfate. After evaporation the product is purifiedby silica gel column chromatography using 5% methanol in dichloromethaneto yield 0.8 mmol.

Preparation of4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoic acid

4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate (100mg) is combined with sodium iodide (63 mg) and acetonitrile (10 mL), andthen chlorotrimethylsilane (120 mL) is slowly added. The mixture isheated at 70° C. for 6 hours, cooled and filtered. The solid product iswashed with water, then ether to yield 51 mg.

Preparation of 2,5-dioxopyrrolidin-1-yl4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate

4-[6-amino-8-methoxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzoate (2mmol) is dissolved in dichloromethane or dioxane (10 mL) and EDC (2mmol) is added. To this solution is added N-hydroxysuccinimide (2 mmol)and resulting mixture is stirred at room temperature for 1 hour. Themixture is taken to dryness in vacuo and the crude product is purifiedby silica gel chromatography to yield 2 mmol of product that is suitablefor conjugation reactions involving primary amines.

Example II

UC-1V150 was covalently coupled to MSA first modified with asuccinimidyl 6-hydrazino-nicotinamide acetone hydrazone (SANH) linker toyield a stable molecule with a characteristically altered UV spectrum.The UC-1V150/MSA conjugate was identified by a UV absorption peak at 342nm due to hydrazone formation, whereas SANH alone absorbed at 322 nm.Quantification of UC-1V150 molecules conjugated per MSA was extrapolatedfrom a standard curve of UC-1V150-SANH (FIG. 1). Consistently theUC-1V150/MSA conjugates were obtained at a ratio of about 5:1. Thebiological studies reported here were done by using 5:1 UC-1V150/MSA.

Modification of MSA with SANH.

200 μL of MSA (25 mg/mL) was mixed with 100 μL of conjugation buffer (1MNaPi, pH=7.2) and 690 μl of PBS. 844 μg of SANH in 10 μL of DFM (40-foldmolar excess to MSA) was added to protein solution (final concentrationof MSA in reaction mixture is 5 mg/mL). After gentle mixing reaction wasproceeded at room temperature for 2 hours. To remove excess of SANH thereaction mixture was loaded on NAP-10 column equilibrated with PBS andmodified MSA was eluted with 1.5 mL of PBS.

Attachment of IV 150 to MSA Modified with SANH.

460 μg of IV 150 dissolved in 10 μL of DMF was added to MSA modifiedwith SANH and the reaction mixture was incubated at RT overnight. Toremove excess of IV 150 the reaction mixture was firstly concentrated to1 mL using micro-spin column (Millipore: BIOMAX 5K) and loaded on NAP-10column as mentioned above.

TLR7 agonists were also conjugated to oligodeoxynucleotides (ODNs)(FIGS. 20-21), a virus (FIGS. 18-19), and to a lipid component which canthen be incorporated into a liposome (FIGS. 24-25).

Synthesis of Spatially Regulated TLR7 Agonist.

Each conjugate is prepared by standard techniques well known inbioconjugation chemistry. Characterization of each by quantitative UV,LC/MS, and PAGE methods determines the “valence” or ratio of TLR agonistto its auxiliary group (macromolecule). From this information, the sizeand shape of the conjugates are easily estimated by modeling techniques.The diversity in size, shape, and valence of the conjugates isintroduced through the selection of the macromolecule, represented inthe structure scheme as R3. For example, when R3 is a dendrimer, such asof the common poly(amidoamine) variety, the number of surface functionalgroups for attachment of the TLR agonist is precisely defined based onthe number of branching points or generations of that particulardendrimer. A first generation (G1) has 8 surface amino groups, a G2 has16, and so on, thus resulting in a high level of control over valenceand size of the conjugates (see FIG. 2). Additionally, some dendrimernanoparticles may contain both a targeting ligand and the TLR7 agonist.The TLR7 agonist-lipid conjugates may also have a variety of “valences”depending on the selection of the lipids. For example, the potentconjugate UV-1V199/L (FIG. 6) was prepared by coupling a carboxyderivative of the TLR7 agonist (UC-1V199) to the ethanolamino group ofthe commercially available dioleanylphosphatidylethanolamine (DOPE).

These lipid conjugates are formulated into various liposomenanoparticles by combining with cholesterol, DOPE, and other lipids toproduce particles having a hydrodynamic diameter of about 100 nm (FIGS.24-25). The hexagons in the figure represent UC-1V199/L and related TLR7agonist with phospholipid tails.

TLR7 agonists and dimers, as well as TLR conjugates have been shown tohave cytokine releasing and/or cytokine activity in vivo as determinedby assays such as those disclosed herein. For instance, imiquimod,bropirimine, UC-1V138, UC-1V136, UC-1V150, UC-1X105, UC-1V199, UC-1W236,UC-1X51, UC-1W247, UC-1X113, UC-1V199/L, UC-1V150/BSA, conjugates ofUC-1V150 with or without a linker and MSA, OVA, virions, and/or ODN,conjugates of UC-1V199 and DOPE, silica, lipid, or irradiated spores,and conjugates of UC-1V1043 and UC-1V1018 with OVA all have shownactivity.

Example III Materials and Methods

Compound Evaluation In Vitro.

The ability of TLR7 conjugates to stimulate and/or to inhibit cytokineproduction is assessed in murine bone marrow derived mononuclear cells(BMDM) that are highly enriched in dendritic cells, as well as in humanperipheral blood mononuclear cells (PBMC) cells. BMDM are plated in 96well plates and treated in triplicate with vehicle or various dosesstarting from 10 μM diluted in 3-fold increments down to picomolarconcentrations. After 24 hours the supernatants are harvested andassayed for up to 30 different cytokines, chemokines and othermediators, using a Luminex bead assay system, and commercially availablereagents. The cytokine/chemokine ELISA results are supplemented withquantitative mRNA expression measurements and with two-dimensionalphosphoprotein analyses, to gain insight into the scope and mechanism oftolerance induction. At the time of supernatant harvest, media arereplaced in the wells with MTT, as a colorimetric assessment of cellsurvival. Human PBMC are isolated from commercial blood packs andtreated similarly.

To assess the trafficking of the TLR agonist-conjugated nanoliposomesand dendrimers, the respective nanoparticles are loaded or modified witha fluorochrome. Subcellular localization is determined microscopically,in some cases in cells that have been treated with inhibitors ofendosomal maturation.

To compare the anti-inflammatory activities of the TLR7 conjugates withdifferent auxiliary groups, BMDM are treated first with the most potentcompounds, at previously determined concentrations that had minimaleffects on pro-inflammatory cytokine stimulation (TNFα, IL-1). After 24hours, the medium is replaced, and the cells are challenged withactivating ligands of different TLR family members (Pam3Cys for TLR2,poly(I:C) for TLR3, LPS for TLR4, flagellin for TLR5, Malp-2 for TRL6,UC-1V150 for TLR7, R848 for TLR7/8, CpG oligonucleotides for TLR9, andthe like) at concentrations that effectively induce cytokine productionin mock treated cells. The cells are assessed by multiplex immunoassay,quantitative PCR and phosphoprotein blotting. To better understand thekinetics of induction and maintenance of tolerance, TLR7conjugate-primed cells are also challenged at different time intervalsand analyzed for the pattern of cytokine production.

Compound Evaluation In Vivo.

Production of bronchoalveolar lavage fluid (BALF) versus systemiccytokines after administration to the airways of mice is assessed.Anesthetized female age-matched C57BL/6 mice are administered nasally(i.n.) orally (p.o.), or intravenously (i.v.), with various amounts ofthe TLR7 conjugates as previously described, or with the liposomes ordendrimers in appropriate vehicles. After recovery and at different timepoints, sera and BALF are collected and analyzed for cytokines andchemokines by Luminex assay. The weights, temperatures, and fluid intakepatterns of the treated animals are recorded, as a clinical surrogatefor a systemic “cytokine syndrome.”

Subsequent experiments assess the ability of the different agents toproduce local and systemic refractoriness (TLR tolerance) to TLRactivation after high dose administration i.n., p.o. or i.v., asdetermined by sera and BALF cytokines High doses of the various TLR7conjugates, which do not induce significant cytokines in vivo, norclinical signs of a cytokine syndrome, are selected. Mice are treatedwith the selected high doses given by the different routes ofadministration, and then challenged with activators of different TLRs atvarious time points. Serum and BALF are collected and analyzed andclinical symptoms are recorded. The anti-inflammatory activities of theconjugates are confirmed with a lethal shock model previously used tostudy LPS and CpG. In this model, Balb/c mice that have been previouslyinjected i.p. with D-galactosamine succumb after systemic challenge withdifferent TLR activators, due to cytokine stimulation and liver damage.Active anti-inflammatory drugs fail to induce clinical symptoms insensitized animals, and will also prevent shock caused by other TLRligands. With a defined endpoint, this model is especially useful fordetermining the kinetics and duration of TLR tolerance.

Example IV Materials and Methods

Mice.

Female C57BL/6 mice (5-6 weeks of age) were obtained from Harlan WestCoast (Germantown, Calif.), and female A/J mice (6-8 weeks of age) werepurchased from The Jackson Laboratories (Bar Harbor, Me.). A/J mice wereused for infection with the Sterne strain of B. anthracis (Kenney etal., J. Infect. Dis., 190:774 (2004)). The mice were bred and maintainedunder standard conditions in the University of California at San DiegoAnimal Facility, which is accredited by the American Association forAccreditation of Laboratory Animal Care. All animal protocols receivedprior approval by the Institutional Review Board. For the H1N1 influenzastudy, female BALB/c mice (16-18 g) were obtained from Charles RiverLaboratories (Wilmington, Mass.) and maintained in the AmericanAssociation for Accreditation of Laboratory Animal Care-accreditedLaboratory Animal Research Center of Utah State University.

In Vitro Stimulation of BMDM.

BMDM were isolated from various strains of mice were seeded in 96-wellplates at a density of 5×10⁴ cells per well. Compounds were added to10-day-old cultures at a final concentration ranging from 0.01 to 10 μMor as otherwise indicated. After 24 hours of incubation, culturesupernatants were collected and assayed for cytokine inductions byeither sandwich ELISA (BD Pharmingen, San Diego, Calif.) or multiplexLuminex (Austin, Tex.) assay using the Beadlyte Mouse MultiCytokinecustomized kit (Upstate, Charlottesville, Va., and eBiosciences, SanDiego, Calif.), according to the manufacturer's instructions.

Administration of Compounds to Mice.

Female age-matched C57BL/6 mice were injected with 100 μL of salinesolution containing UC-1V150 or UC-1V150/MSA, each containing theequivalent of 0.38-38 nmol of the pharmacore via the tail vein. Forintrapulmonary administration, mice were anesthetized with i.p. Avertinsolution and shaved around the neck area. The trachea were exposed witha small incision and injected with 50 μL of saline solution containingvarious amounts of UC-1V150/MSA or the unconjugated drug. After recoveryand at different time points, serum and BALF were collected and analyzedfor IL-6, IL-12p40, IFN-γ, RANTES, and MCP-1 by Luminex assay. In otherexperiments, mice were anesthetized with an intramuscularketamine/xylene solution and administered the same amount ofUC-1V150/MSA in i.t. doses of 50 μl or i.n. doses of 20 μL. Becausesimilar cytokine levels were observed in the BALF 24 hours afteradministration by either method, the more convenient i.n. route was usedin infectious model studies.

Infection of A/J Mice with B. anthracis Spores.

Spores were prepared from the Sterne strain of B. anthracis (pXO1⁺pXO2⁻)as previously described (Sabet et al., FEMS Immunol. Med. Microbiol.,47:369 (2006); Guidi-Rontani et al., Mol. Microbiol., 42:931 (2001)).Purified spores were stored in PBS at 1×10⁸ to 4×10⁸ cfu/mL at 4° C.Before infection, the spores were heated to 65° C. for 30 minutes toinitiate germination. A/J mice were anesthetized intramuscularly withketamine/xylene solution and administered i.n. with 0.75 nmol ofUC-1V150 or UC-1V150/MSA per mouse 1 day before anthrax infection.Control mice received saline only or saline containing MSA at equivalentamounts as in UC-1V150/MSA. Infection was carried out i.n. with 2×10⁵ to8×10⁵ spores of B. anthracis in a 20 μL volume. Survival was observedfor 13 days, because the majority of the saline-treated mice died within3-6 days. Results were obtained from eight mice per group.

Infection of Balb/c Mice with Influenza Virus.

Influenza A/New Caledonia/20/99 (H1N1) virus was obtained from theCenters for Disease Control and Prevention (Atlanta, Ga.). The virus waspropagated twice in Madin Darby canine kidney (MDCK) cells, furtherpassaged 7 times in mice to make it virulent followed by another passagein cell culture to amplify it. Mice were anesthetized i.p. with ketamine(100 mg/kg) and infected i.n. with virus at approximately 10^(5.0) cellculture infectious doses per mouse in a 50 μL inoculum volume. A singleintranasal dose of 75 μL in either saline alone or containingUC-1V150/MSA to 5 nmole per mouse was given 24 hours prior to virusexposure. Ten infected mice per treated group and 20 placebo controlanimals were followed for survival for 21 days.

Statistics.

Cytokine levels were compared by the Mann Whitney U-test with p≦0.05 todetermine statistical significance. Kaplan-Meier survival curves and logrank tests were performed using GraphPad Prism software version 4.0c(San Diego, Calif.) to compare differences in survival.

Results

Potent In Vitro and In Vivo Cytokine Release in Response to UC-1V150/MSAConjugates.

Incubation of bone-marrow-derived macrophages (BMDM) with UC-1V150 alonestimulated cytokine release (FIG. 10). When conjugated to MSA, similaror higher levels of cytokines were detected with a 10-fold lowerequivalent concentration of the TLR7 agonist. Experiments with TLRtransformants, performed as described previously, confirmed thatUC-1V150, similar to the compound lacking the aldehyde modification(UC-1V136), was a specific TLR7 agonist (Lee et al., Proc. Natl. Acad.Sci. USA, 103:1828 (2006)). After i.v. injection into mice, UC-1V150induced serum cytokine levels that peaked at about 2 hours afterinjection and then quickly declined to near background levels (data notshown). Comparison of the cytokine production profiles of UC-1V150versus the UC-1V150/MSA 2 hours after i.v. injection at various dosagesdemonstrated that the MSA conjugate enhanced the potency by 10- to100-fold (FIG. 11). Sera from saline or MSA control groups revealedlittle or no detectable cytokine levels (data not shown).

UC-1V150/MSA Conjugates Provide Prolonged and Localized PulmonaryActivity.

To ensure adequate delivery of the TLR7 agonists to the respiratorysystem, the drugs were initially directly into the trachea. Substantialcytokine induction was found in bronchial alveolar lavage fluid (BALF)extracted from mice treated intratracheally (i.t.) with UC-1V150/MSA,whereas serum cytokines were very low and near background levels in thesame animals (FIG. 12). In marked contrast, similar levels of cytokinewere observed in both BALF and sera of mice injected i.t. withsmall-molecule TLR7 agonists, which sometimes induced behavioralchanges, such as hair standing on end and shivering, suggestive of acytokine syndrome (Table 2). Subsequent studies with UC-1V150 showedthat intranasal (i.n.) delivery also induced selective cytokineproduction in the BALF, probably due to drug aspiration. Accordingly,i.n. administration was used to evaluate the UC-1V150 conjugates in twoinfectious animal models of pneumonitis. Mice pretreated i.n. withUC-1V150/MSA one day before infection with B. anthracis spores had anextended mean survival of 7.5 days compared with 5 days in control mice(P<0.025) (FIG. 14A). In contrast, no significant difference wasobserved in mice treated with either saline, the equivalent amount ofMSA, or with UC-1V150 alone. These data confirmed that the UC-1V150conjugate, but not the free drug, had intrapulmonary immunotherapeuticactivity. Thus, conjugation of the TLR7 agonist to MSA enhanced itspotency and reduced its toxicity after local delivery to the respiratorytract.

In another study, BALB/c mice were pretreated i.n. with the UC-1V150/MSAconjugate 1 day before influenza virus infection (H1N1 strain). The meansurvival of the treated mice was extended to 11.5 days compared with 7days in untreated controls (P<0.0001) (FIG. 14B). Together these resultssuggest that conjugation of the TLR7 agonist to MSA enhanced its potencyand reduced its toxicity after local delivery to the respiratory tract.

UC-1V150/MSA was administered i.n. prior to anthrax infection followedby treatment with ciprofloxacin (25 mg/kg) on day 4. Placebo treatmentfollowed by ciprofloxacin treatment resulted in about 15-25% survival,while treatment with a conjugate and ciprofloxacin resulted in about 90%survival. Thus, the conjugate is particularly useful as a coadjuvantwith an anthrax vaccine.

Discussion

The compound UC-1V150 is one of the most potent and versatile syntheticsmall-molecule TLR7 ligands yet discovered because (i) it is active atnanomolar concentrations; (ii) it can be coupled to a variety ofmacromolecules with enhancement of activity in some cases; and (iii) itspharmacokinetic properties can be changed by modification of theauxiliary groups. The TLR7-protein conjugate UC-1V150/MSA wascharacterized as having approximately five small molecules covalentlylinked to each MSA protein molecule. The conjugate retained TLR7 agonistactivity and indeed was both more potent and had a longer duration ofaction, compared with the free monomeric drug. Moreover, this conjugatecould be delivered effectively to the respiratory system by i.n. or i.t.administration. Drug delivery by i.n. proved to be effective in a mousemodel of a bacterial infection. When considering delivery to therespiratory system, a potentially important advantage of preparing theTLR7 agonists as conjugates of macromolecules is that systemic sideeffects may be avoided by confining the immunostimulatory activity tothe local mucosal environment.

The macromolecular conjugate would be expected to be absorbed into thesystemic circulation more slowly than the free drug and, indeed, may beavidly scavenged by resident macrophages and dendritic cells expressingTLR7. Accordingly, the conjugate should mitigate the type of severe sideeffects that have been associated with systemic delivery of TLR7/8agonists. The UC-1V150/MSA conjugate may also provide beneficialimmunotherapeutic activity when administered to mucosal sites, such asthe genitourinary and gastrointestinal tracts, for the control ofinfectious, allergic, or malignant diseases. The macromolecular carrierof the TLR7 agonist may also provide an improved approach for selectivedelivery of the immunotherapeutic to a specific organ or tissue. Forexample, the lipid conjugates of UC-1V150 can be incorporated intoliposomes of different size and composition, whereas protein conjugatesof the TLR7 agonist may target different dendritic cell subsets.Differences in the intracellular trafficking of the UC-1V150 conjugatemay induce distinct patterns of cytokine production, analogous to theeffects observed with TLR9-activating oligonucleotides (Rothenfusser etal., Hum. Immunol., 63:111 (2002)).

One potential problem that has been observed with drugs conjugated toproteins is the development of antibodies against thelow-molecular-weight hapten-like portion of the molecule. However,UC-1V150, unlike the TLR7/8 vaccine conjugates studied earlier, has asimple adenine-like structure that is unlikely to inducehypersensitivity reactions. Indeed, anti-UC-1V150 antibodies were notobserved after administration of the protein conjugates, except afterrepeated administration of a keyhole limpet hemocyanin carrier incomplete Freud's adjuvant (unpublished data).

New agents for the prevention and treatment of influenza virusinfections are being sought, particularly with the spread of highlypathogenic strains from Asia. Morbidity and mortality from commonlycirculating strains is high each year. Treatment of the infection can beaccomplished by approved antiviral drugs, which are moderately effectiveif started early. Enhancement of the immune system is also beinginvestigated as a strategy that could accelerate protective antiviralresponses, especially in immune compromised hosts. It is possible thatsystemic immune activation via TLR signaling does not create a localcytokine and chemokine gradient required to mobilize immune cells to thesite of infection. In support of this hypothesis, the unconjugatedUC-1V150, which is rapidly absorbed through the mucosa, failed toprotect mice from B. anthracis infection, whereas the UC-1V150 conjugatewas effective.

B. anthracis has become an agent of bioterrorism. A rapid responseagainst microbial pathogens is critical for effective biodefense. Ingeneral an antibody or cellular immune response may protect againstthese pathogens; however, generating these protective responses quicklyrequires prior exposure to specific antigens for each organism. Althoughit is known that influenza virus engages TLR7 (Barchet et al., Eur. J.Immunol., 35:360 (2005)), bacterial anthrax most likely can engage TLR2,TLR4, and TLR9. In addition to being a common signaling intermediary forthe TLRs, MyD88 has also been shown to be necessary for resistance toinfection in a mouse model of anthrax (Hughes et al., Infect. Immun.,73:7535 (2005)). Because the UC-1V150 conjugate works effectively as anadjuvant against infections that use different pathways, it can beapplied as a biodefense strategy that would not need be specific to theantigens of a particular microbe and that would be useful in mixed aswell as single agent attacks.

Example V

There is no known SA vaccine that is potent enough or that can actquickly enough to prevent SA infections in “at-risk” patients prior tohospitalization. A single injection of a potent TLR7 agonist and killedgram-positive bacteria, e.g., SA, or a subunit thereof, may boostprotective immunity to the bacteria within one week of administration.The injection may include, for example, 1) a TLR7 agonist such UC-IV199conjugated directly to free amino groups on killed gram-positivebacteria, 2) a TLR7 agonist such as UC-IV199 conjugated to albumin incombination with killed gram-positive bacteria, 3) a TLR7 agonist suchas UC-IV199 conjugated to a recombinant gram-positive bacterial protein,or 4) a TLR7 agonist such as UC-IV199 conjugated to gram-positivebacterial polysaccharides (e.g., via a linker known to the art, such asthat used the StaphVax®).

As described hereinabove, a TLR7 agonist was conjugated to lethallyirradiated spores of the Sterne vaccine strain of Bacillus anthracis(BA). Like SA, BA is a gram-positive bacteria. Compared to spores alone,the conjugated bacterium was a potent activator of mouse bone marrowderived macrophages (BMDM) as measured by cytokine (IL-12 and IL-6)secretion. In another experiment, a single injection into mice oflethally irradiated spores of the Sterne strain of BA, mixed with a TLR7agonist conjugated to mouse albumin (MSA), protected the animals againstlethal intra-pulmonary BA challenge given only six days later. Incontrast, injection of the animals with BA spores alone, or with BA plusa conventional adjuvant, cholera toxin (CT), did not protect theanimals. Thus, a TLR7-agonist albumin/irradiated spore vaccine inducedprotective immunity to Bacillus anthracis within 6 days. This rapidityof response in a naïve animal was totally unexpected. The same vaccinetechnology will likely protect humans from hospital-acquired SAinfection.

All publications, patents, and patent documents cited in thespecification are incorporated by reference herein, as thoughindividually incorporated by reference. In the case of anyinconsistencies, the present disclosure, including any definitionstherein will prevail. The invention has been described with reference tovarious specific and preferred embodiments and techniques. However, itshould be understood that many variations and modifications may be madewhile remaining within the spirit and scope of the invention.

What is claimed is:
 1. A composition comprising a compound of formula A:

or a pharmaceutically acceptable salt thereof, wherein X¹ is —O—; R¹ ishydrogen, (C₁-C₁₀)alkyl, or, (C₁-C₁₀)alkyl substituted with(C₁-C₆)alkoxy; X² is C(O); and R³ is a phosphatidyl ethanolaminecomprising two C₁₄-C₂₂ carboxylic acid groups each having one, two,three, or four sites of unsaturation, epoxidation, hydroxylation, or acombination thereof, each at a location of the respective carboxylicacid group carbon chain.
 2. The composition of claim 1 wherein X²-R³ isbonded to a carbon atom para to the methylene carbon bonded to theheteroaryl nitrogen atom.
 3. The composition of claim 1, wherein the twocarboxylic acid groups of the phosphatidyl ethanolamine of the compoundof Formula A are the same.
 4. The composition of claim 1, wherein eitheror both carboxylic acid groups of the phosphatidyl ethanolamine of thecompound of Formula A is a C₁₇ carboxylic acid group with a site ofunsaturation at C8-C9.
 5. The composition of claim 1, wherein either orboth carboxylic acid groups of the phosphatidyl ethanolamine of thecompound of Formula A is a C₁₈ carboxylic acid group with a site ofunsaturation at C9-C10.
 6. The composition of claim 1, wherein R¹ of thecompound of Formula A is a (C₁-C₁₀)alkyl substituted with (C₁-C₆)alkoxy.7. The composition of claim 1, wherein for the compound of Formula A, R¹is —(CH₂)₂—OCH₃.
 8. The composition of claim 1, which further comprisesa liposome.
 9. A composition comprising an amount of a compound ofFormula A of claim 1 effective as an adjuvant.
 10. A vaccine comprisingan antigen and a compound of Formula A of claim
 1. 11. The compositionof claim 1, wherein the two carboxylic acid groups of phosphatidylethanolamine of the compound of Formula A are different.